EA047973B1 - COMBINATION THERAPY CONTAINING ANTI-CD19 DRUG CONJUGATE AND PI3K INHIBITOR - Google Patents

Description

Previous applications

This application claims priority from the following two applications: (1) UK application GB1908233.8, filed on 10 June 2019; and (2) UK application GB1908234.6, filed on 10 June 2019.

Field of technology

The present invention relates to combination therapy options for the treatment of pathological conditions such as cancer. In particular, the present invention relates to combination therapy options comprising treatment with a CD19 antibody-drug conjugate (anti-CD19 ADC) and a phosphoinositide 3-kinase (PI3K) inhibitor or a secondary agent.

State of the art

Antibody therapy.

Antibody therapies have been developed to target patients with cancer, immunological and angiogenic disorders (Carter, P. (2006) Nature Reviews Immunology 6:343-357). The use of antibody-drug conjugates (ADCs), i.e. immunoconjugates, for local delivery of cytotoxic or cytostatic agents, i.e. drugs for the destruction or inhibition of tumor cells, in the treatment of cancer is aimed at the delivery of the drug component to tumors and their intracellular accumulation, while systemic administration of these unconjugated drugs can lead to unacceptable levels of toxicity to healthy cells (Xie et al (2006) Expert. Opin. Biol. Ther. 6(3): 281–291; Kovtun et al (2006) Cancer Res. 66(6): 3214–3121; Law et al (2006) Cancer Res. 66(4): 2328–2337; Wu et al (2005) Nature Biotech. 23(9):1137–1145; Lambert J. (2005) Current Opinion. in Pharmacol. 5: 543-549; Hamann P (2005) Expert Opin. Ther. Patents 15(9): 1087-1103; Payne, G. (2003) Cancer Cell 3: 207-212; Trail et al (2003) Cancer Immunol. Immunother. 52: 328-337; Syngos and Epenetos (1999) Anticancer Research 19:605-614).

CD19.

CD19 is a 95 kDa membrane receptor that is expressed early in B cell differentiation and continues to be expressed until B cells enter terminal differentiation (Pezzutto et al. (1987) J. Immunol 138:2793; Tedder et al (1994) Immunol Today 15:437). The extracellular domain of CD19 contains two immunoglobulin (Ig)-like domains of the C2 type separated by a smaller, potentially disulfide-linked domain. The cytoplasmic domain of CD19 is unique in its structure but is highly conserved between human, mouse, and guinea pig (Fujimoto et al., (1998) Semin Immunol. 10: 267). CD19 is part of a protein complex found on the cell surface of B lymphocytes. The protein complex includes CD19, CD21 (complement receptor type 2), CD81 (TAPA-1), and CD225 (Leu-13) (Fujimoto, see above).

CD19 is an important regulator of transmembrane signaling in B cells. Increases or decreases in CD19 surface density affect B cell development and function, leading to diseases such as autoimmune diseases or hypogammaglobulinemia. The CD19 complex enhances the B cell response to antigen in vivo by cross-linking two separate signal transduction complexes found on B cell membranes. The two signal transduction complexes, membrane IgM and CD19, activate phospholipase C (PLC) by distinct mechanisms. Cross-linking of CD19 and B cell receptors reduces the number of IgM molecules required for PLC activation. CD19 also functions as a specialized adaptor protein for the amplification of Arc family kinases (Hasegawa et al., (2001) J Immunol 167:3190).

CD19 binding has been shown to both enhance and inhibit B cell activation and proliferation, depending on the degree of cross-linking (Tedder, 1994, Immunol. Today 15:437). CD19 is expressed in more than 90% of B cell lymphomas and is thought to influence lymphoma growth in vitro and in vivo.

Therapeutic use of anti-CD19 ADC.

The efficacy of an antibody-drug conjugate comprising an antibody to CD19 (anti-CD19-ADC) in the treatment of, for example, cancer has been established - see, for example, WO2014/057117 and WO2016/166298.

Research is ongoing to further improve the efficacy, tolerability, and clinical utility of anti-CD19 ADCs. To this end, the present inventors have identified clinically advantageous combination therapy options in which anti-CD19 ADCs are administered in combination with at least one PI3K inhibitor or secondary agent.

The essence of the invention

The inventors have found that administering to an individual a combination of an anti-CD19 ADC and a PI3K inhibitor or secondary agent results in unexpected clinical benefits. The inventors have further determined that administering an anti-CD19 ADC to an individual who has either been treated or is being treated with a PI3K inhibitor or secondary agent results in a synergistic increase in treatment efficacy.

Accordingly, the first aspect of this description proposes a method for selecting an individual,

- 1 047973 suitable for treatment with an aHTu-CD19 ADC, wherein said individual is selected for treatment with an anti-CD19 ADC if the individual has received or is receiving treatment with a PI3K inhibitor or a secondary agent. The individual may be selected for treatment if he or she is refractory to treatment or further treatment with a PI3K inhibitor or a secondary agent.

In another aspect of the disclosure, there is provided a method of treating a disorder in an individual, said method comprising selecting an individual suitable for treatment by the method of the first aspect, and then administering to said individual an effective amount of an anti-CD19 ADC. The method of treatment may further comprise administering a PI3K inhibitor or a secondary agent in combination with an anti-CD19 ADC.

In another aspect, the description provides a method for treating a disorder in an individual, wherein said method comprises administering to the individual an effective amount of an anti-CD19 ADC and a PI3K inhibitor or secondary agent. The individual may be selected for treatment according to the method of the first aspect.

The disorder in question may be a proliferative disorder, such as cancer such as non-Hodgkin's lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphoid lymphoma (CLL), and marginal zone B-cell lymphoma (MZBCL), and leukemia such as hairy cell leukemia (HCL), hairy cell variant leukemia (HCL-v), and acute lymphoblastic leukemia (ALL), such as Philadelphia chromosome-positive ALL (Ph+ ALL) or Philadelphia chromosome-negative ALL (Ph- ALL).

Ahtu-CD19 ADC may be ADCX19 described herein.

The PI3K inhibitor may be idelalisib, copanlisib, duvelisib, taselisib, buparlisib, alpelisib, umbralisib, dactolisib and voxtalisib. Preferably, the PI3K inhibitor is idelalisib or copanlisib.

The secondary agent may be:

(a) Bendamustine;

(b) Lenalidomide;

(c) a proteasome inhibitor such as bortezomib, carfilzomib, ixazomib, oprozomib, and salinosporamide A; or (d) a PARP inhibitor such as olaparib, CEP-9722, BMN-673/talazoparib, rucaparib, iniparib/SAR24-550/BSI-201, veliparib (ABT-888), niraparib/MK-4827, BGB-290, 3-aminobenzamide, and E7016.

An individual may be a human. An individual may have cancer or have been determined to have cancer. An individual may have or have been determined to have CD19+ cancer or CD19+ tumor-associated non-tumor cells, such as infiltrating CD19+ B cells.

The Ahtu-CD19 ADC in the disclosed methods may be administered prior to the PI3K inhibitor or secondary agent, concurrently with the PI3K inhibitor or secondary agent, or subsequent to the PI3K inhibitor or secondary agent. The disclosed methods may include administering to the individual an additional chemotherapeutic agent.

In another aspect of the disclosure, there are provided an anti-CD19 ADC or a composition comprising an anti-CD19 ADC for use in a method of treatment as described herein.

In one aspect of the disclosure, there is provided a PI3K inhibitor or secondary agent, or a composition comprising a PI3K inhibitor or secondary agent, for use in the treatment method described herein.

In a further aspect, this disclosure provides the use of an anti-CD19 ADC, PI3K inhibitor, or secondary agent in the manufacture of a medicament for treating a disorder in an individual, wherein said treatment comprises a method of treatment as described herein.

In another aspect of the disclosure, a first composition comprising an anti-CD19 ADC is provided for use in a method of treating a disorder in an individual, wherein said treatment comprises administering the first composition in combination with a second composition comprising a PI3K inhibitor or a secondary agent.

This aspect also provides a first composition comprising a PI3K inhibitor or a secondary agent for use in a method of treating a disorder in an individual, wherein the treatment comprises administering the first composition in combination with a second composition comprising an anti-CD19 ADC.

The disorder in question may be a proliferative disorder, such as cancer such as non-Hodgkin's lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphoid lymphoma (CLL), and marginal zone B-cell lymphoma (MZBCL), and leukemia such as hairy cell leukemia (HCL), hairy cell variant leukemia (HCL-v), and acute lymphoblastic leukemia (ALL), such as Philadelphia chromosome-positive ALL (Ph+ ALL) or Philadelphia chromosome-negative ALL (Ph- ALL).

Ahtu-CD19 ADC may be ADCX19 described herein.

- 2 047973

The PI3K inhibitor may be idelalisib, copanlisib, duvelisib, taselisib, buparlisib, alpelisib, umbralisib, dactolisib and voxtalisib. Preferably, the PI3K inhibitor is idelalisib or copanlisib.

The secondary agent may be:

(a) Bendamustine;

(b) Lenalidomide;

(c) a proteasome inhibitor such as bortezomib, carfilzomib, ixazomib, oprozomib, and salinosporamide A; or (d) a PARP inhibitor such as olaparib, CEP-9722, BMN-673/talazoparib, rucaparib, iniparib/SAR24-550/BSI-201, veliparib (ABT-888), niraparib/MK-4827, BGB-290, 3-aminobenzamide, and E7016.

An individual may be a human. An individual may have cancer or have been determined to have cancer. An individual may have or have been determined to have CD19+ cancer or CD19+ tumor-associated non-tumor cells, such as infiltrating CD19+ B cells.

The first composition may be administered before, simultaneously with, or after the second composition. The treatment may include administering an additional chemotherapeutic agent to the individual.

In a further aspect of the present invention there is provided the use of an anti-CD19-ADC in the manufacture of a medicament for treating a disorder in an individual, wherein the medicament comprises an hhtu-CD19 ADC, and wherein the treatment comprises administering the medicament in combination with a composition comprising a PI3K inhibitor or a secondary agent.

This aspect also provides the use of a PI3K inhibitor or secondary agent in the manufacture of a medicament for treating a disorder in an individual, wherein the medicament comprises a PI3K inhibitor or secondary agent, and wherein the treatment comprises administering the medicament in combination with a composition comprising an anti-CD 19 ADC.

The disorder in question may be a proliferative disorder, such as cancer such as non-Hodgkin's lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphoid lymphoma (CLL), and marginal zone B-cell lymphoma (MZBCL), and leukemia such as hairy cell leukemia (HCL), hairy cell variant leukemia (HCL-v), and acute lymphoblastic leukemia (ALL), such as Philadelphia chromosome-positive ALL (Ph+ ALL) or Philadelphia chromosome-negative ALL (Ph- ALL).

Ahtu-CD19 ADC may be ADCX19 described herein.

The PI3K inhibitor may be idelalisib, copanlisib, duvelisib, taselisib, buparlisib, alpelisib, umbralisib, dactolisib and voxtalisib. Preferably, the PI3K inhibitor is idelalisib or copanlisib.

The secondary agent may be:

(a) Bendamustine;

(b) Lenalidomide;

(c) a proteasome inhibitor such as bortezomib, carfilzomib, ixazomib, oprozomib, and salinosporamide A; or (d) a PARP inhibitor such as olaparib, CEP-9722, BMN-673/talazoparib, rucaparib, iniparib/SAR24-550/BSI-201, veliparib (ABT-888), nipanarib/MK-4827, BGB-290, 3-aminobenzamide, and E7016.

An individual may be a human. An individual may have cancer or have been determined to have cancer. An individual may have or have been determined to have CD19+ cancer or CD19+ tumor-associated non-tumor cells, such as infiltrating CD19+ B cells.

The drug may be administered prior to the composition, simultaneously with the composition, or after the composition. Treatment may include administering an additional chemotherapeutic agent to the individual.

In another aspect of the present invention there is provided a kit comprising:

the first drug containing hhtu-CD19 ADC;

a package insert containing instructions for administering the first medicinal product in accordance with the method of treatment described herein. The kit may further comprise a second medicinal product comprising a PI3K inhibitor or a secondary agent.

In another aspect of the present invention, there is provided a kit comprising: a first medicament comprising an anti-CD19 ADC;

a second medicament comprising a PI3K inhibitor or secondary agent and, optionally, a package insert containing instructions for administering the first medicament to an individual in combination with the second medicament for treating a disorder.

In this aspect, a kit is also provided comprising a medicinal product comprising an anti-CD19 ADC and a package insert comprising instructions for administering the medicinal product to an individual.

- 3 047973 duumu in combination with a composition containing a PI3K inhibitor or a secondary agent, for the treatment of a disorder.

Additionally, this aspect provides a kit comprising a medicament comprising a PI3K inhibitor or secondary agent and a package insert comprising instructions for administering the medicament to an individual in combination with a composition comprising an anti-CD19 ADC for treating a disorder.

The disorder in question may be a proliferative disorder, such as cancer such as non-Hodgkin's lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphoid lymphoma (CLL), and marginal zone B-cell lymphoma (MZBCL), and leukemia such as hairy cell leukemia (HCL), hairy cell variant leukemia (HCL-v), and acute lymphoblastic leukemia (ALL), such as Philadelphia chromosome-positive ALL (Ph+ ALL) or Philadelphia chromosome-negative ALL (Ph- ALL).

Ahtu-CD19 ADC may be ADCX19 described herein.

The PI3K inhibitor may be idelalisib, copanlisib, duvelisib, taselisib, buparlisib, alpelisib, umbralisib, dactolisib and voxtalisib. Preferably, the PI3K inhibitor is idelalisib or copanlisib.

The secondary agent may be:

(a) Bendamustine;

(b) Lenalidomide;

(c) a proteasome inhibitor such as bortezomib, carfilzomib, ixazomib, oprozomib, and salinosporamide A; or (d) a PARP inhibitor such as olaparib, CEP-9722, BMN-673/talazoparib, rucaparib, iniparib/SAR24-550/BSI-201, veliparib (ABT-888), niraparib/MK-4827, BGB-290, 3-aminobenzamide, and E7016.

An individual may be a human. An individual may have cancer or have been determined to have cancer. An individual may have or have been determined to have CD19+ cancer or CD19+ tumor-associated non-tumor cells, such as infiltrating CD19+ B cells.

A drug or composition comprising an anti-CD19 ADC may be administered prior to a drug or composition comprising a PI3K inhibitor or secondary agent, concurrently with a drug or composition comprising a PI3K inhibitor or secondary agent, or subsequent to a drug or composition comprising a PI3K inhibitor or secondary agent. Treatment may include administering an additional chemotherapeutic agent to the individual.

In yet another further aspect, the present invention provides a composition of an anti-CD19 ADC and a PI3K inhibitor or secondary agent.

In this aspect, the present invention also provides a method of treating a disorder in an individual, said method comprising administering to the individual an effective amount of a composition comprising an anti-CD19 ADC and a PI3K inhibitor or secondary agent.

In this aspect, the present invention also provides a composition comprising an anti-CD19 ADC and a PI3K inhibitor or secondary agent for use in a method of treating a disorder in an individual.

In this aspect, the present invention also provides the use of a composition comprising an anti-CD19 ADC and a PI3K inhibitor or secondary agent in the manufacture of a medicament for treating a disorder in an individual.

In this aspect, the present invention also provides a kit comprising a composition comprising an anti-CD19 ADC and a PI3K inhibitor or secondary agent, and a set of instructions for administering the medicament to an individual for treating a disorder.

The disorder in question may be a proliferative disorder, such as cancer such as non-Hodgkin's lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphoid lymphoma (CLL), and marginal zone B-cell lymphoma (MZBCL), and leukemia such as hairy cell leukemia (HCL), hairy cell variant leukemia (HCL-v), and acute lymphoblastic leukemia (ALL), such as Philadelphia chromosome-positive ALL (Ph+ ALL) or Philadelphia chromosome-negative ALL (Ph- ALL).

Ahtu-CD19-ADC may be ADCX19 as described herein.

The PI3K inhibitor may be idelalisib, copanlisib, duvelisib, taselisib, buparlisib, alpelisib, umbralisib, dactolisib and voxtalisib. Preferably, the PI3K inhibitor is idelalisib or copanlisib.

The secondary agent may be:

(a) Bendamustine;

(b) Lenalidomide;

(c) a proteasome inhibitor such as bortezomib, carfilzomib, ixazomib, oprozomib, and salinosporamide A; or

- 4 047973 (d) PARP inhibitor such as olaparib, CEP-9722, BMN-673/talazoparib, rucaparib, uHunapu6/SAR24-550/BSI-201, veliparib (ABT-888), niraparib/MK-4827, BGB-290, 3-aminobenzamide, and E7016.

An individual may be a human. An individual may have cancer or have been determined to have cancer. An individual may have or have been determined to have CD19+ cancer or CD19+ tumor-associated non-tumor cells, such as infiltrating CD19+ B cells.

Treatment may involve administering an additional chemotherapeutic agent to the individual.

Detailed description of the essence of the invention

Antibody-drug conjugates (ADCs).

The present invention relates to improved efficacy of combinations of an ADC and a PI3K inhibitor or secondary agent.

The ADC can deliver a drug to a target site. The target site is preferably a population of proliferative cells. The antibody is an antibody to an antigen present in a population of proliferative cells. In one aspect, the antigen is absent or present at a reduced level in a population of non-proliferative cells compared to the amount of antigen present in a population of proliferative cells, such as a population of tumor cells.

The ADC may comprise a linker that can be cleaved to release the drug at a target site. The drug may be a compound selected from RelA, RelB, RelC, RelD, or RelE. Thus, the conjugate can be used to selectively deliver a RelA, RelB, RelC, RelD, or RelE compound to a target site.

The linker can be cleaved by an enzyme present at the target site.

The present invention relates in particular to treatment with the anti-CD19 ADC described in WO2014/057117 and as described herein.

anti-CJ19 ADC.

As used herein, the terms anti-CD19 ADC and CD19-ADC refer to an ADC in which the antibody component is an antibody to CD19. The term PBD-ADC refers to an ADC in which the drug component is a pyrrolobenzodiazepine (PBD) active payload. The term anti-CD19-ADC refers to an ADC in which the antibody component is an antibody to CD19 and the drug component is a PBD active payload.

An ADC may comprise a conjugate of the formula L-( ^DL ) _P , where DL has the formula I or II:

Where:

L is an antibody (Ab), which is an antibody that binds to CD19;

when a double bond is present between C2' and C3', R ¹² is selected from the group consisting of: (ia) a C _5-10 aryl group optionally substituted with one or more substituents selected from the group consisting of: halogen, nitro, cyano, ether, carboxy, ester, C _1-7 alkyl, C _3-7 heterocyclyl and bis-oxy-C _1-3 alkylene;

(ib) C _1-5 saturated aliphatic alkyl;

(ic) C _3-6 saturated cycloalkyl;

R ²²

R ²³ (id), wherein each of R ²¹ , R ²² and R ²³ is independently selected from H, C _1-3 saturated alkyl, C _2-3 alkenyl, C _2-3 alkynyl and cyclopropyl, wherein the total number of carbon atoms in the R ¹² group is no more than 5;

- 5 047973 (ie)

' where is one of

R ^25a and R ^25b represent H and the other is selected from: phenyl, wherein phenyl is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl; and

(if) ^R24 .-4-,,. . .

' where R is selected from: H; C _1-3 saturated alkyl; C _2-3 alkenyl; C _2-3 alkynyl; cyclopropyl; phenyl which is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl;

where there is a single bond between C2' and C3',

R ^26a

R ¹² is 'where R ^26a and R ^26b are independently selected from H, F, C _1-4 saturated alkyl C _2-3 alkenyl, wherein the alkyl and alkenyl groups are optionally substituted with a group selected from C _1-4 alkylamido and C _1-4 alkyl ester or, where one of R ^26a and R ^26b is H, the other is selected from nitrile and C _1-4 alkyl ester;

R ⁶ and R ⁹ are independently selected from H, R, OH, OR, SH, SR, NH ₂ , NHR, NRR', nitro, Me ₃ Sn and halide;

wherein R and R' are independently selected from optionally substituted C _1-12 alkyl, C _3-20 heterocyclyl and C5-20 aryl groups;

R ⁷ is selected from H, R, OH, OR, SH, SR, NH ₂ , NHR, NHRR', nitro, Me ₃ Sn and halogen;

R is a C _3-12 alkyl group, the chain of which may be interrupted by one or more heteroatoms, for example O, S, NRN ² (where RN ² is H or C _1-4 alkyl), and/or aromatic rings, for example benzene or pyridine;

Y and Y' are selected from O, S or NH;

R ⁶ , R ⁷ , R ⁹ are selected from the same groups as R ⁶ , R ⁷ and R ^{9 ,} respectively;

[Formula I].

R ^L1 is a linker for coupling with an antibody (Ab);

R ^11a is selected from OH, ORA, where R ^a is C _1-4 alkyl, and SOZM, where z is 2 or 3, and M is a monovalent pharmaceutically acceptable cation;

R ²⁰ and R ²¹ either together form a double bond between the nitrogen and carbon atoms to which they are bonded, or;

R ²⁰ is selected from H and R ^C , where R ^C is a blocking group;

R ²¹ is selected from OH, ORA and SOZM;

where there is a double bond between C2 and C3, R ² is selected from the group consisting of:

(ia) a C _5-10 aryl group optionally substituted with one or more substituents selected from the group consisting of: halogen, nitro, cyano, ether, carboxy, ester, C _1-7 alkyl, C _3-7 heterocyclyl and bis-oxy-C _1-3 alkylene;

(ib) C _1-5 saturated aliphatic alkyl;

(ic) C _3-6 saturated cycloalkyl;

R ¹²

R ¹³ (id) ^R > wherein each of R ¹¹ , R ¹² and R ¹³ is independently selected from H, C _1-3 saturated alkyl; C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, wherein the total number of carbon atoms in the R ² group is no more than 5;

R ^15b

(ie)

R ^15a ' wherein one of R ^15a and R ^15b is H and the other is selected from: phenyl, wherein phenyl is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl; and

(if)

R ¹⁴ 14 g where R ¹⁴ is selected from: H; C _1-3 saturated alkyl; C _2-3 alkenyl; C _2-3 alkynyl;

cyclopropyl; phenyl which is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl;

- 6 047973 where there is a single bond between C2 and C3,

AU ^6a

R ^16b

R ² is > where R ^16a and R ^16b are independently selected from H, F, C _1-4 saturated alkyl; C _2-3 alkenyl, wherein the alkyl and alkenyl groups are optionally substituted with a group selected from C _1-4 alkylamido and C _1-4 alkyl ester or, where one of R ^16a and R ^16b is H, the other is selected from nitrile and C _1-4 alkyl ester.

[Formula II].

R ²² has formula IIIa, formula IIIb or formula IIIc:

XL 1„A~ 2-X III» (a) Q «la where A is a C _5-7 aryl group and one of (i) Q ¹ is a single bond and Q ² is selected from a single bond and -Z-(CH ₂ ) _n -, where Z is selected from a single bond, O, S and NH, and n is from 1 to 3; or (ii) Q ¹ is -CH=CH- and Q ² is a single bond;

R ^C2

Where;

R ^C1 , R ^C2 and R ^C3 are independently selected from H and unsubstituted C _1-2 alkyl;

ts (c) where Q is selected from O-RL ² , S-RL ² and NRN-RL ² , and RN is selected from H, methyl and ethyl,

X is selected from the group consisting of: O-RL ² , S-RL ² , CO2-RL ² , CO-RL ² , NH-C(=O)-RL ² , NHNH-RL ² , CONHNH-RL ² ', |—\i—r ^L2 ' f/ \i—r ^L2 '>> NRNRL ² , where RN is selected from the group consisting of H and C _1-4 alkyl;

RL ² is a linker for coupling with an antibody (Ab);

R ¹⁰ and R ¹¹ either together form a double bond between the nitrogen and carbon atoms to which they are bonded, or;

R ¹⁰ is H and R ¹¹ is selected from OH, ORA and SO _Z M;

R ³⁰ and R ³¹ either together form a double bond between the nitrogen and carbon atoms to which they are bonded, or;

R ³⁰ is H and R ³¹ is selected from OH, ORA and SO _Z M.

In some embodiments, L-RL ¹ or L-RL ² is a group:

C ^Ab X /L!?O

O where the asterisk indicates the point of attachment to the PBD, Ab is an antibody, ^L1 is a cleavable linker, A is a linking group connecting ^L1 to the antibody, ^L2 is a covalent bond or together with -OC(=O)- forms a self-cleavable linker.

In some of these embodiments, L ¹ is cleaved by an enzyme.

Such ADCs have previously been shown to be useful in the treatment of CD19 expressing cancers (see, for example, WO2014/057117, which is incorporated herein by reference in its entirety).

The term anti-CD19-ADC may include any embodiment described in WO2014/057117. In particular, in preferred embodiments, the ADC may have the chemical structure:

- 7 047973

where Ab is CD19 antibody and DAR is from 1 to 8.

The antibody may comprise a VH domain having a sequence according to any of SEQ ID NO. 1, 2, 3, 4, 5 or 6, optionally further comprising a VL domain having a sequence according to any of SEQ ID NO. 7, 8, 9, 10, 11 or 12.

In some aspects, the anti-CD19-ADC antibody component is an antibody comprising: VH and VL domains, respectively, having the sequences: SEQ ID NO: 1 and SEQ ID NO: 7, SEQ ID NO. 2 and SEQ ID NO: 8, SEQ ID NO. 3 and SEQ ID NO: 9, SEQ ID NO. 4 and SEQ ID NO: 10, SEQ ID NO. 5 and SEQ ID NO: 11, or SEQ ID NO. 6 and SEQ ID NO: 12.

In preferred embodiments, the antibody comprises a VH domain having the sequence according to SEQ ID NO. 2. In preferred embodiments, the antibody comprises a VL domain having the sequence according to SEQ ID NO. 8.

In preferred embodiments, the antibody comprises a VH domain and a VL domain, wherein the VH domain has the sequence of SEQ ID NO. 2 and the VL domain has the sequence of SEQ ID NO. 8.

The VH and VL domain(s) can pair to form the antibody's antigen-binding site, which binds CD19.

In preferred embodiments, the antibody is an intact antibody comprising a VH domain and a VL domain, wherein the VH and VL domains have the sequences of SEQ ID NO: 2 and SEQ ID NO: 8.

In some embodiments, the antibody is an antibody comprising a heavy chain having the sequence of SEQ ID NO. 13 and a light chain having the sequence of SEQ ID NO. 14.

In some embodiments, the antibody is a fully human IgG1 monoclonal antibody, preferably IgG1,K.

In some embodiments, the antibody is the RB4v1.2 antibody described in WO2014/057117.

In one aspect, the antibody is an antibody described herein that has been modified (or further modified) as described below. In some embodiments, the antibody is a humanized, deimmunized, or resurfaced version of an antibody described herein.

The most preferred anti-CD19 ADC for use with aspects of this disclosure is ADCxl9, as described herein below.

A second preferred anti-CD19 ADC for use with aspects of this disclosure is ADCT-402 (Loncastuximab tesirine).

ADCx19.

ADCx19 is an antibody-drug conjugate consisting of a humanized anti-human CD19 antibody attached to a pyrrolobenzodiazepine (PBD) warhead via a cleavable linker. The mechanism of action of ADCX19 is dependent on CD19 binding. An antibody specific for CD19 targets the antibody-drug conjugate (ADC) to cells expressing CD19. Upon binding, the ADC is internalized and transported to the lysosome, where the protease-sensitive linker is cleaved and the free PBD dimer is released within the target cell. The released PBD dimer inhibits transcription selectively by directly inhibiting RNA polymerase or inhibiting the interaction of associated transcription factors. The PBD dimer forms covalent cross-links that do not distort the DNA double helix and are not recognized by nucleotide excision repair factors, allowing for a longer effective period (Hartley 2011).

- 8 047973

It has the chemical structure:

The Ab is the RB4v1.2 antibody (the antibody with the VH and VL sequences of SEQ ID NO. 2 and SEQ ID NO: 8, respectively). This antibody is synthesized as described in WO2014/057117 (RB4v1.2E) and typically has a DAR (drug to antibody ratio) of 2 ± 0.3.

CD19 binding.

As used herein, the term "binds CD19" means that the antibody binds CD19 with a higher affinity than a non-specific partner such as bovine serum albumin (BSA, Genbank Accession No. CAA76847, Version No. CAA76847.1 GI: 3336842, Entry Update Date: January 7, 2011, 2:30 PM). In some embodiments, the antibody binds CD19 with an association constant (K _a ) that is at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, ¹⁰⁴ , ¹⁰⁵ , or ¹⁰⁶ times greater than the association constant of the antibody for BSA when measured under physiological conditions. Antibodies of the present invention can bind CD19 with high affinity. For example, in some embodiments, an antibody can bind CD19 with a KD equal to or less than about 10 ^-6 M, such as 1 x 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 , 10 ^-13 or 10 ^-14

In some embodiments, the CD19 polypeptide corresponds to Genbank accession number NP_001171569, version no. NP_001171569.1 GI: 296010921, entry update date: September 10, 2012, 12:43. In one embodiment, the nucleic acid encoding the CD19 polypeptide corresponds to Genbank accession number NM_001178098, version no. NM_001178098.1 GI: 296010920, entry update date: September 10, 2012, 12:43. In some embodiments, the CD19 polypeptide corresponds to Uniprot/Swiss-Prot accession number P15391.

PI3K inhibitors.

The vertebrate class I PI 3-kinase family of enzymes comprises four distinct protein species of approximately 110 kDa (p110a, p110β, ρ110δ, and ρ110γ). Most structural features and substrate specificities are common to all class I enzymes (Rameh and Cantley, 1999; Fry, 2001; Katso et al, 2001). In vitro, all class I PI 3-kinases are able to phosphorylate PtdIns to PtdIns(3)P, PtdIns(4)P to PtdIns(3,4)P2, and PtdIns(4,5)P2 to PtdIns(3,4,5)P3, with PtdIns(4,5)P2 considered the preferred lipid substrate in vivo. Class I PI 3-kinases are primarily found in the cytosol in resting cells but, when stimulated, are recruited to membranes through interactions with receptors or adaptor proteins. They are thought to function primarily at the plasma membrane, but there have been reports of class I PI 3-kinases associated with vesicular and nuclear membranes (Rameh and Cantley, 1999; Fry, 2001; Katso et al., 2001). The cellular roles of class I PI 3-kinases are diverse, with evidence linking them to cell size, motility, survival, and proliferation in response to numerous signaling systems in many different cell types (Fry, 2001; Katso et al., 2001). The class I family is divided into two groups based on their regulatory partners and mechanisms of activation.

Although PI3K was initially characterized two decades ago through its association with oncogenes and activated RTKs (reviewed in Zhao JJ et al., 2006), its association with human cancer was only established in the late 1990s when the tumor suppressor PTEN was demonstrated to function as a GN-lipid phosphatase. Recent comprehensive genomic analyses of cancer have demonstrated that many components of the PI3K pathway are frequently mutated or altered in common human cancers, highlighting the importance of this pathway in cancer (see Wood LD, et al. Science. 2007; Sun Y, et al. Science. 2004).

The term phosphoinositide 3-kinase inhibitors (PI3K inhibitors) is used herein to refer to any agent that specifically binds to and/or inhibits the biological activity of PI3K.

As used herein, the term "specifically binds PI3K" means that the agent binds PI3K with higher affinity than a non-specific partner such as bovine serum albumin (BSA, Genbank Accession No. CAA76847, Release No. CAA76847.1 GI: 3336842, Entry Update Date: January 7, 2011, 2:30 p.m.). In some embodiments, the agent binds PI3K with a

- 9 047 973 association constant (K _a ) that is at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 ⁴ , 10 ⁵ or 10 ⁶ times higher than the association constant of the agent for BSA when measured under physiological conditions. The agents can bind PI3K with high affinity. For example, in some embodiments, the agent can bind PI3K with a KD equal to or less than about 10 ^-6 M, such as 1 x 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 - ⁹ , 10 ^-10 , 10 ^-11 , 10 ^-12 , 10 ^-13 or 10 - ¹⁴ .

PI3K inhibitors suitable for use in this description include:

a) copanlisib.

i. CAS number ^ 1032568-63-0, (see http://www.cas.org/content/chemical-substances/faqs).

ii. DrugBank link ^DB12483, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^ WI6V529FZ9, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula I: copanlisib, 2-amino-and-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl]pyrimidin-5- carboxamide

b) idelalisib.

i. CAS number ^ 870281-82-6, (see http://www.cas.org/content/chemical-substances/faqs).

ii. DragBank reference ^ DB09054, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^YG57I8T5M0, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula II: idelalisib, 5-fluoro-3-phenyl-2-[(^)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone. c) duvelisib.

i. CAS number ^ 1201438-56-3, (see http://www.cas.org/content/chemical-substances/faqs).

ii. DragBank reference ^ DB11952, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^610V23S0JI, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

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Formula III: duvelisib, 8-chloro-2-phenyl-3-[(^)-1-(3H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone. d) Taselisib.

i. CAS number ^ 1282512-48-4, (see http://www.cas.org/content/chemical-substances/faqs).

ii. Unique Ingredient Identifier (UNII) ^ L08J2O299M, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula IV: taselisib, 2-{4-[2-(1-isopropyl-3-methyl-III-1,2,4-triazol-5-yl)-5,6-dihydroimidazo[1,2-d][ 1,4] benzoxazepin-9-yl] -1 H-pyrazol-1 -yl} -2-methylpropanamide.

e) Buparlisib.

i. CAS number ^ 944396-07-0, (see http://www.cas.org/content/chemical-substances/faqs).

ii. DrugBank reference ^ DB11666, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^ 0ZM2Z182GD, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula V: buparlisib, 5-[2,6-bis(morpholin-4-yl)pyrimidin-4-yl]-4-(trifluoromethyl)pyridin-2 amine.

f) Alpelisib.

i. CAS number ^ 1217486-61-7, (see http://www.cas.org/content/chemical-substances/faqs).

ii. Link to DrugBank^ (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^08W5N2C97Q, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

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Formula VI: alpelisib, (^)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide.

g) Umbralisib.

i. CAS number ^ 1532533-67-7, (see http: //www. cas.org/content/chemical-substances/faqs).

ii. DragBank reference ^ DB14989, (see https://www.drugbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^38073MQB2A, (see http://www. fda. gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default. htm).

Formula VII: umbralisib, (^)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one.

h) Dactolisib.

i. CAS number ^ 915019-65-7, (see http: //www. cas.org/content/chemical-substances/faqs).

ii. DrugBank link ^DB11651, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^RUJ6Z9Y0DT, (see http://www. fda. gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default. htm).

Formula VIII: dactolisib, 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-Shimidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile.

i) Voxtalisib.

i. CAS number ^ 934493-76-2, (see http: //www. cas.org/content/chemical-substances/faqs).

ii. DragBank link ^ DB12400, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^CVL1685GPH, (see http://www. fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

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Formula IX: 2-amino-8-ethyl-4-methyl-6-(1H-pyrazol-5-yl)-7H,8H-pyrido[2,3^]pyrimidin-7-one.

Preferably, the PI3K inhibitor is idelalisib or copanlisib. Most preferably, the PI3K inhibitor is idelalisib.

Secondary agents.

The recent development of agents that enhance antitumor immunity is rapidly changing the treatment approach for a wide range of cancers. However, these treatment options are not effective for all cancer types, and responses to treatment are often short-lived, with little or no benefit in many patients. The prevailing wisdom in the oncology field is that only combinations of immunotherapy drugs with other treatment options will ultimately cure cancer patients.

The ADC is well tolerated and active in a variety of cancer types and is likely to be one component of combination therapy that increases response rates and duration of treatment. The aim of this invention is to combine the ADC with a secondary agent.

The secondary agent, as described herein, may be an immuno-oncology (IO) agent.

Immuno-oncology (IO) drugs, which are cancer therapies that use the body's immune system to help fight cancer, have been shown to produce more durable anti-tumor responses. There are different types of IOs, including but not limited to PD1 inhibitors, PD-L1 inhibitors, CLTL4 inhibitors, GITR agonists, and OX40 agonists. Because a significant proportion of patients are not cured by single-agent immunotherapy and eventually relapse, combination therapy with alternative IO agents or other therapeutic modalities is necessary (see KS Peggs et al. 2009, Clinical and Experimental Immunology, 157: 9–19 [doi:10.1111/j.l365-2249.2009.03912.x]; DM Pardoll 2012 [doi:10.1038/nrc3239]).

Immunogenic cell death (ICD) is a specific form of cell death that stimulates an immune response against dead cell antigens (released by dying cells) and is considered one of the best ways to induce an adaptive immune response and improve the efficacy of anticancer treatments. This process is often suboptimal and requires the use of combinatorial strategies aimed at restoring the full immunogenicity of cell death for therapeutic purposes. There are several anticancer agents that can induce ICD, such as various anthracyclines (including doxorubicin, epirubicin, and idarubicin), alkylating agents (including oxaliplatin and cyclophosphamide), mitoxantrone, a topoisomerase II inhibitor, and the proteasome inhibitor bortezomib.

Antibody-drug conjugates, including those with an active PBD moiety, may be particularly suitable as combination partners because they are more targeted than traditional chemotherapy and are expected to provide enhanced antigen presentation to infiltrating T cells, as has been demonstrated for auristatin-based ADCs.

Thus, combining ADC with IO provides a dual benefit: on the one hand, ADC will directly kill the tumor expressing the target, providing immediate antitumor activity, and on the other hand, the immunogenic cell death induced by ADC-mediated cell killing can enhance a stronger and more robust adaptive immune response compared to when IO is administered as monotherapy.

To demonstrate that anti-CD19 ADCs function synergistically with the secondary agent, a panel of CD19(+) cell lines were co-treated with a range of concentrations of both anti-CD19 ADCs and the secondary agent. As a negative control, the same panel of cell lines was treated with a range of concentrations of the secondary agent or a range of concentrations of anti-CD19 ADCs and vehicle. Following incubation, two parameters were measured: the amount of surface CD19 (determined by flow cytometry) and the in vitro cytotoxicity of the combinations (determined by MTS assays). To determine cytotoxicity, cell viability was measured by adding MTS to the well and incubating for 4 hours at 37°C. The percentage of cell viability compared to the untreated control was calculated. Cytotoxic synergy was calculated by converting cell viability data to the proportion of dead cells and calculating the additivity index using the CalcuSyn analysis program.

The secondary agent may be:

(a) Bendamustine;

- 13 047973 (b) Lenalidomide;

(c) a proteasome inhibitor such as bortezomib, carfilzomib, ixazomib, oprozomib, and salinosporamide A; or (d) a PARP inhibitor such as olaparib, CEP-9722, BMN-673/talazoparib, rucaparib, iniparib/SAR24-550/BSI-201, veliparib (ABT-888), niraparib/MK-4827, BGB-290, 3-aminobenzamide, and E7016.

Each of these classes of secondary agents is described in more detail below.

Bendamustine.

Bendamustine is a bifunctional mechlorethamine derivative capable of forming electrophilic alkyl groups that covalently bind to other molecules. Due to this function as an alkylating agent, bendamustine provides intra- and inter-strand cross-links between DNA bases, which leads to cell death. The drug is active against both active and immobile cells.

Bendamustine is indicated for the treatment of chronic lymphocytic leukemia (CLL) and indolent B-cell non-Hodgkin lymphoma (NHL) that has progressed during or within six months of treatment with rituximab or a rituximab-containing regimen.

1. CAS number ^ 16506-27-7, (see http://www. cas. org/content/chemical-sub stances/faqs).

ii. Unique Ingredient Identifier (UNII) ^9266D9P3PQ, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifi erUNII/default.htm).

Formula X: Bendamustine, 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid. Lenalidomide.

Lenalidomide is a thalidomide analogue with enhanced immunomodulatory and antiangiogenic effects that does not cause most of the typical adverse events associated with thalidomide.

In myelodysplastic syndromes (MDS), this drug has been used primarily in the low- and intermediate-risk IPSS 1 setting. Several clinical studies have demonstrated its ability to induce both erythroid and cytogenetic responses in these disease groups. In a clinical trial of patients with the chromosomal abnormality del(5q), treatment with lenalidomide resulted in red blood cell (RBC) transfusion independence in 67% of patients. Moreover, 45% of patients achieved complete cytogenetic remission and 28% achieved minor cytogenetic remission. This outcome was independent of the complexity of the karyotype. Lenalidomide can also induce durable remission in del(5q) patients with elevated medullary blast counts. Among patients without del(5q), 43% of patients with confirmed low- and intermediate-risk IPSS 1 achieved transfusion independence or at least a 50% reduction in RBC transfusion levels compared with pretreatment levels.

Adverse events are common but treatable and include neutropenia and thrombocytopenia, pruritus, rash, diarrhea, and others. Lenalidomide may prove to be an important component of therapeutics for MDS. Combination therapy with cytokines, demethylating agents, tyrosine kinase inhibitors, or chemotherapy is being studied and may show additional benefit in both low- and high-risk MDS (see Giagounidis et al., Ther Clin Risk Manag. 2007 Aug; 3(4): 553-562).

i. CAS number ^ 191732-72-6, (see http://www.cas.org/content/chemical-substances/faqs).

ii. Unique Ingredient Identifier (UNII) ^ F0P408N6V4, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifi erUNII/default.htm).

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Formula XI: Lenalidomide, 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione.

Proteasome inhibitors.

The proteasome is a large protein complex responsible for the degradation of intracellular proteins, a process that requires metabolic energy. Polymerization of ubiquitin, a key molecule known to function in concert with the proteasome, serves as a degradation signal for multiple target proteins; protein degradation is initiated by the covalent attachment of a chain consisting of multiple copies of ubiquitin (more than four ubiquitin molecules) through the concerted actions of a network of proteins including the E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-ligating) enzymes. The polymerized ubiquitin chain acts as a signal that delivers target proteins to the proteasome, where the substrate is proteolytically cleaved. Numerous enzymes (e.g., 2 E1 proteins, ~30 E2 proteins, and over 500 different E3 species in humans) are mobilized for precise protein selection by this cascade system. The E3 protein repertoire is highly diverse because each E3 enzyme typically selectively recognizes a protein substrate for ubiquitination (see Tanaka 2009, Proc Jpn Acad Ser B Phys Biol Sci. 2009 Jan; 85(1): 12–36 and references therein).

The ubiquitin-proteasome system (UPS) controls nearly all major cellular processes, such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, quality control, and protein development, by degrading short-lived regulatory or structurally aberrant proteins.

These protein regulatory processes are also important in cancer, and thus the proteasome is an important regulator of carcinogenesis. Cancer pathologies include many cells that, according to the cancer stem cell theory, originate from a small percentage of cancer stem cells, also called tumor-initiating cells. These cells constitute the subsets that have the capacity to propagate all the various cancer pathologies and to repopulate tumors after cytostatic therapy. The proteasome plays a role in cellular processes in cancer stem cells, but has been found to have lesser function in them compared to other cancer cells. In particular, the proteasome has been reported to play a role in proliferation and pluripotency, which is a defining characteristic of cancer cells and cancer stem cells (see Voutsadakis et al., Tumor Biology, Mar. 2017).

The term proteasome inhibitors is used herein to refer to any agent that specifically binds to and/or inhibits the biological activity of a component of the proteasome.

As used herein, the term "specifically binds a component of the proteasome" means that the agent binds the component of the proteasome with a higher affinity than a non-specific partner, such as bovine serum albumin (BSA, Genbank Accession No. CAA76847, Version No. CAA76847.1 GI: 3336842, Entry Update Date: January 7, 2011, 2:30 PM). In some embodiments, the agent binds the component of the proteasome with an association constant ( _Ka ) that is at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, ¹⁰⁴ , ¹⁰⁵ , or ¹⁰⁶ times higher than the association constant of the agent for BSA when measured under physiological conditions. These agents may bind a component of the proteasome with high affinity. For example, in some embodiments, the agent may bind a component of the proteasome with a KD equal to or less than about 10 ^-6 M, such as 1 x 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 , 10 ^-13 or 10 ^-14 .

Proteasome inhibitors suitable for use herein include:

a) bortezomib.

i. CAS number ^ 179324-69-7, (see http://www. cas. org/content/chemical-substances/faqs).

ii. DrugBank link ^DB00188, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^69G8BD63PP, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

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Formula XII: bortezomib, [(^)-3-methyl-1-({^)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid.

b) carfilzomib.

i. CAS number ^ 868540-17-4, (see http://www.cas.org/content/chemical-substances/faqs).

ii. DrugBank reference ^ DB08889, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^72X6E3J5AR, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula XIII: carfilzomib, (2S)-4-methyl-N-[(2S)-1-[[(2S)-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1oxopentan- 2-yl]amino]-1-oxo-3-phenylpropan-2-yl]-2-[[^)-2-[(2-morpholin-4-ylacetyl)amino]-4phenylbutanoyl]amino]pentanamide.

c) Ixazomib.

i. CAS number ^ 1072833-77-2, (see http://www.cas.org/content/chemical-substances/faqs).

ii. DragBank reference ^ DB09570, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^ 71050168A2, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula XIV: ixazomib, til]glycinamide.

N-(2,5-dichlorobenzoyl)-i-[(^)-1-(dihydroxyboryl)-3-methylbu-

d) Oprozomib.

i. CAS number ^ 935888-69-0, (see http://www.cas.org/content/chemical-substances/faqs).

ii. DragBank reference ^ DB11991, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^ MZ37792Y8J, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

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Formula XV: oprosomib, N-[(VS)-3-\ieToi<cu-1-[[(2S)-3-\ieTOi<cu-1-[[(2S)-1-[(2R)-2 -\ieTu _B ioi<cupaii-2yl]-1-oxo-3-phenylpropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]-2-methyl- 1,3-thiazole-5-carboxamide.

f) Salinosporamide A.

i. CAS number ^ 437742-34-2, (see http://www.cas.org/content/chemical-substances/faqs).

ii. DrugBank reference ^ DB11762, (see https://www.dragbank.ca/).

iii. Unique Ingredient Identifier (UNII) ^703P9YDP7F, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula XVI: salinosporamide A, (4R,5S)-4-(2-chloroethyl)-1-((1S)-cyclohex-2-enyl(hydroxy)methyl)5-methyl-6-oxa-2-azabicyclo [3.2 .0]heptane-3,7-dione.

PARP inhibitors.

Poly(adenosine diphosphate [ADP]) ribose polymerase (PARP) is a family of enzymes involved in a wide range of cellular functions, including DNA transcription, DNA damage response, maintenance of genomic stability, cell cycle regulation, and cell death. PARP-1 is the most abundant and best characterized protein in this group. In oncology, its integral role in the repair of DNA single-strand breaks (SSBs) via the base excision repair (BER) pathway has been the focus of attention, and several PARP-1 inhibitors (PARPi) (including, but not limited to, olaparib, CEP-9722, talazoparib, rucaparib, iniparib, veliparib, and niraparib) have been developed and are undergoing clinical trials. In cancer therapy, PARPi acts primarily by preventing the repair of DNA damage, which ultimately leads to cell death.

PARP consists of four domains of interest: the DNA-binding domain, the caspase-cleaved domain, the auto-modification domain, and the catalytic domain. The DNA-binding domain consists of two zinc finger motifs. In the presence of damaged DNA (excised base pairs), the DNA-binding domain will bind DNA and induce a conformational shift. It has been demonstrated that this binding occurs independently of the other domains. This is an integral part of the programmed cell death model based on the inhibition of PARP cleavage by caspase. The auto-modification domain is responsible for the release of the protein from DNA after catalysis. In addition, it plays an important role in cleavage-induced inactivation.

PARP is located in the cell nucleus. Its primary role is to detect and initiate an immediate cellular response to metabolic, chemical, or radiation-induced single-strand DNA breaks (SSBs) by signaling the enzymatic machinery involved in SSB repair. Once PARP detects an SSB, it binds to the DNA, undergoes structural changes, and initiates the synthesis of a polymer chain of adenosine diphosphate-ribose (poly(ADP-ribose) or PAR), which acts as a signal for other DNA-stopping enzymes. Target enzymes include DNA ligase III (LigIII), DNA polymerase beta (pole), and scaffold proteins such as X-ray cross-complementation gene 1 (XRCC1). Once repaired, PAR chains are degraded by poly(ADP-ribose) glycohydrolase (PARG).

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NAD+ is required as a substrate for the formation of ADP-ribose monomers. It is thought that excessive PARP activation may deplete cellular NAD+ stores and cause progressive ATP depletion and necrotic cell death because glucose oxidation is inhibited. However, it has recently been proposed that inhibition of hexokinase activity leads to glycolytic defects (see Andrabi, PNAS 2014). Note that PARP is inactivated by caspase-3 cleavage during programmed cell death.

PARP enzymes are important for a number of cellular functions, including the expression of inflammatory genes: PARP1 is required for the induction of ICAM-1 gene expression by smooth muscle cells in response to TNF.

PBDs are a class of natural antitumor antibiotics found in Streptomyces. PBD dimers exert their cytotoxic mechanism of action by cross-linking two DNA strands, leading to blockade of replication and tumor cell death. Importantly, the cross-links formed by PBD dimers are relatively non-distorting to DNA structure, making them elusive to DNA repair mechanisms, which are often impaired in human tumors, unlike normal tissues.

The advantage of combining PBD-based ADCs with PARPi (including, but not limited to, olaparib, CEP-9722, talazoparib, rucaparib, iniparib, veliparib, and niraparib) is that repair of DNA damaged by PBD dimers is blocked by PARP inhibition, resulting in accumulation of DNA damage that causes cancer cell death.

To demonstrate that treatment of solid tumor-derived cell lines with PBD-based ADCs and PARPi exerts additive or synergistic antitumor effects, a panel of solid tumor-derived cell lines will be treated with a range of concentrations of each ADC and PARPi. Following incubation, the in vitro cytotoxicity of the combinations (as determined by the CellTiter-Glo® or MTS assays) will be measured. Cytotoxic synergy was calculated by converting cell viability data to proportion of dead cells and calculating the additivity index using the CalcuSyn analysis program.

A PARP inhibitor means that any chemical compound or biological molecule reduces the activity of PARP.

In order to study the degree of inhibition of, for example, PARP activity, samples or assays containing, for example, said protein, gene, cell or organism are treated with a potential activating or inhibitory agent and compared with control samples treated with an inactive control molecule. The control samples are assigned a relative activity value of 100%. Inhibition is achieved when the activity value relative to the control sample is 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, typically 50% or less, more often 45% or less, most often 40% or less, preferably 35% or less, more preferably 30% or less, even more preferably 25% or less and most preferably less than 20%.

Specific PARPi suitable for use in the present invention include:

a) Olaparib.

i. CAS number ^ 763113-22-0.

ii. NCBI Pubchem reference ^23725625.

iii. Unique Ingredient Identifier (UNII) ^ WOH1JD9AR8.

Formula XVII, olaparib: 4-[(3-[(4-cyclopropylcarbonyl)piperazin-1-yl]carbonyl)-4-fluorophenyl]methyl(2H)phthalazin-1-one.

b) SER-9722.

1. CAS number ^ 916574-83-9, (see http://www.cas.org/content/chemical-substances/faqs).

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Formula XVIII, CEP-9722: 11-methoxy-2-((4-methylpiperazin-1-yl)methyl)-4,5,6,7-tetrahydro-1Hcyclopenta[a]pyrrolo[3,4-c]carbazole-1,3(2H)-dione.

c) BMN-673/talazoparib.

i. CAS number ^ 1207456-01-6, (see http://www.cas.org/content/chemical-substances/faqs).

ii. Unique Ingredient Identifier (UNII) ^ 9QHX048FRV.

Formula XIX, talazoparib: (8S, 9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triαzol-5-yl)2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one.

d) Rukaparib.

i. CAS number ^ 283173-50-2, (see http://www.cas.org/content/chemical-substances/faqs).

ii. NCBI Pubchem reference ^9931954, (see https://pubchem.ncbi.nlm.nih.gov/).

iii. Unique Ingredient Identifier (UNII) ^ 8237F3U7EH, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula XX, Rucaparib: 8-fluoro-2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3^]indol-6-one. .

f) iniparib/SAR24-550/BSI-201.

i. CAS number ^ 160003-66-7, (see http://www.cas.org/content/chemical-substances/faqs).

ii. NCBI Pubchem reference ^9796068, (see https://pubchem.ncbi.nlm.nih.gov/).

iii. Unique Ingredient Identifier (UNII)^2ZWI7KHK8F, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

- 19 047973

Formula XXI, Iniparib: 4-iodo-3-nitrobenzamide.

f) Veliparib (AVT-888).

i. CAS number ^ 912444-00-9, (see http://www.cas.org/content/chemical-substances/faqs).

ii. NCBI Pubchem reference ^11960529, (see https://pubchem.ncbi.nlm.nih.gov/).

iii. Unique Ingredient Identifier (UNII) ^01O4K0631N, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula XXII, Veliparib: 2-(^)-2-methylpyrrolidin-2-yl)-III-benzimidazole-4-carboxamide.

g) Niraparib/MK-4827.

i. CAS number ^ 1038915-60-4, (see http://www.cas.org/content/chemical-substances/faqs).

ii. NCBI Pubchem reference ^24958200, (see https://pubchem.ncbi.nlm.nih.gov/).

iii. Unique Ingredient Identifier (UNII) ^ HMC2H89N35, (see http://www.fda.gov/ForIndustry/DataStandards/SubstanceRegistrationSystem-UniqueIngredientIdentifierUNII/default.htm).

Formula XXIII, Niraparib: 2-[4-[^)-3-piperidyl]phenyl]indazole-7-carboxamide. h) BGB-290.

1. CAS number ^ 1820833-75-7, (see http://www.cas.org/content/chemical-substances/faqs).

i) 3-aminobenzamide.

i. CAS number ^ 3544-24-9, (see http://www.cas.org/content/chemical-substances/faqs).

ii. Reference NCBI Pubchem^1645, (see https://pubchem.ncbi.nlm.nih.gov/).

Formula XXIV: 3-aminobenzamide.

j) E7016.

i. CAS number ^ 902128-92-1, (see http://www.cas.org/content/chemical-substances/faqs).

Formula XXV, E706: Benzopyrano(4,3,2-de)phthalazin-3(2H)-one, 10-((4-hydroxy-1-piperidinyl)methyl)Preferably, the secondary agent is olaparib or bendamustine.

- 20 047973

Useful properties of the described combinations.

Both anti-CD19 ADC and PI3K inhibitor or secondary agent, when used as a single agent, have demonstrated clinical benefit, for example in the treatment of cancer. However, as described herein, the combination of anti-CD19 ADC and PI3K inhibitor or secondary agent is expected to provide one or more of the following advantages compared to treatment with anti-CD19 ADC or PI3K inhibitor or secondary agent alone:

1) effective treatment of a wider range of malignant neoplasms;

2) effective treatment of resistant or refractory forms of disorders such as cancer, and individuals with disorders such as cancer who have relapsed after a period of remission;

3) increased response rate to treatment; and/or

4) increased duration of treatment.

Effective treatment of a broader range of malignancies in the context of this description means that a complete response is observed after treatment with the combination in a broader range of recognized malignancy types. Thus, a complete response is observed in malignancy types not previously reported, with a complete response to either the anti-CD19 ADC alone, or the PI3K inhibitor alone, or a secondary agent.

Effective treatment of resistant, refractory or relapsed forms in the context of this description means that after treatment with the combination, a complete response is observed in individuals who are either partially or completely resistant or refractory to treatment with either the anti-CD19 ADC alone, or the PI3K inhibitor alone, or a secondary agent (e.g., individuals who do not respond or only partially respond to treatment with a single agent, or individuals with relapsed disease). In some embodiments, a complete response after treatment with the anti-CD19 ADC/PI3K inhibitor combination or a secondary agent is observed in at least 10% of individuals who are either partially or completely resistant or refractory to treatment with the anti-CD19 ADC or PI3K inhibitor or a secondary agent alone. In some embodiments, a complete response following treatment with an anti-CD19 ADC/PI3K inhibitor combination or a secondary agent is observed in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of individuals who are partially or completely resistant or refractory to treatment with an anti-CD19 ADC or PI3K inhibitor or a secondary agent alone.

An increased response rate to treatment, as used herein, means that a complete response is observed in a greater proportion of individuals following treatment with the combination than is observed following treatment with either the anti-CD19 ADC alone, the PI3K inhibitor alone, or a secondary agent. In some embodiments, a complete response following treatment with the anti-CD19 ADC/PI3K inhibitor combination or a secondary agent is observed in at least 10% of the individuals treated. In some embodiments, a complete response following treatment with an anti-CD19 ADC/PI3K inhibitor or secondary agent combination is observed in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of treated individuals.

Extended duration of treatment as used herein means that the median duration of complete response in individuals receiving the combination is longer than in individuals who achieved a complete response after treatment with only the anti-CD19 ADC or only the PI3K inhibitor or secondary agent. In some embodiments, the median duration of complete response after treatment with the anti-CD19 ADC/PI3K inhibitor or secondary agent combination is at least 6 months. In some embodiments, the median duration of complete response following treatment with an anti-CD19 ADC/PI3K inhibitor combination or secondary agent is at least 12 months, at least 18 months, at least 24 months, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 15 years, or at least 20 years.

In the context of this document, the term complete response is used to indicate the absence of any clinical signs of disease in an individual. The data can be assessed using appropriate methodology in the art, such as CT or PET scanning, or biopsy, where appropriate. The number of doses required to achieve a complete response can be one, two, three, four, five, ten or more. In some embodiments, individuals achieve a complete response no more than a year after the first dose, such as no more than 6 months, no more than 3 months, no more than a month, no more than two weeks, or no more than a week after the first dose.

Disorders that can be treated.

The combination therapies described in this document include those that are useful

- 21 047973 for anticancer activity. In particular, in certain aspects of the therapy, an antibody is included conjugated, i.e., covalently attached by a linker, to a PBD drug moiety, i.e., a toxin. When the drug is not conjugated to the antibody, the PBD drug moiety has a cytotoxic effect. Thus, the biological activity of the PBD drug moiety is modulated by conjugation with the antibody. The antibody-drug conjugates (ADCs) of the present invention selectively deliver an effective dose of a cytotoxic agent to tumor tissue, due to which higher selectivity, i.e., a lower effective dose, can be achieved.

Thus, in one aspect, the present invention provides combination therapy options comprising administering an anti-CD19 ADC that binds CD19 for use in therapy, wherein said method comprises selecting a subject based on expression of a target protein.

In one aspect, the present invention provides a combination therapy with a label that indicates that the therapy is suitable for use in a subject that is determined to be suitable for such use. The label may indicate that the therapy is suitable for use in a subject with CD19 expression, such as CD19 overexpression. The label may indicate that the subject has a particular type of cancer.

The cancer may be a lymphoma, such as non-Hodgkin lymphoma. The label may state that the subject has CD19+ lymphoma.

In a further aspect, a combination therapy as described herein is also provided for use in the treatment of a proliferative disease. Another aspect of this disclosure relates to the use of a conjugate compound in the manufacture of a medicament for the treatment of a proliferative disease.

One skilled in the art can readily determine whether a candidate combination therapy treats a proliferative condition for any particular cell type. For example, assays that are useful for assessing the activity of a particular compound are described below.

The combination therapies described herein may be used to treat a proliferative disease. The term proliferative disease refers to unwanted or uncontrolled cellular proliferation of excess or abnormal cells that is undesirable, such as neoplastic or hyperplastic growth, both in vitro and in vivo.

Examples of proliferative conditions include, but are not limited to, benign, premalignant, and malignant cellular proliferation, including but not limited to neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancer (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g., connective tissue), and atherosclerosis. Cancers of interest include, but are not limited to, leukemias and ovarian cancer.

Any cell type can be treated, including but not limited to lung, gastrointestinal tract (including, for example, intestine, colon), breast, ovary, prostate, liver (hepatocytes), kidney (renal), bladder, pancreas, brain, and skin.

Proliferative disorders of particular interest include, but are not limited to, non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), marginal zone B-cell lymphoma (MZBL), and leukemias such as hairy cell leukemia (HCL), variant hairy cell leukemia (VHL-B), acute lymphoblastic leukemia (ALL) such as Philadelphia chromosome positive ALL (Ph+ ALL) or Philadelphia chromosome negative ALL (Ph- ALL). [Fielding A., Haematologica. 2010 Jan; 95(1): 8-12].

It is contemplated that the combination therapies of the present invention may be used to treat a variety of diseases or disorders, for example, characterized by tumor antigen overexpression. Typical conditions or hyperproliferative disorders include benign or malignant tumors; leukemia, hematological and lymphoid malignancies. Others include neuronal, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stromal, blastocoele, inflammatory, angiogenic and immunological, including autoimmune disorders and graft versus host disease (GVHD).

Typically, the disease or disorder to be treated is a hyperproliferative disease, such as cancer. Examples of cancer to be treated according to the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., squamous cell epithelial carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer,

- 22 047973 Peritoneal cancer, hepatocellular cancer, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

Autoimmune diseases that may benefit from combination therapies include rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune diseases of the gastrointestinal tract and liver (such as, for example, inflammatory bowel diseases (e.g. ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitides (such as, for example, ANCA-associated vasculitis including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteritis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opsomeclonal syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease and autoimmune polyneuropathies), renal dysfunction (such as, for example, glomerulonephritis, Goodpasture's syndrome and Berger's disease), autoimmune dermatological diseases (such as, for example, psoriasis, urticaria, urticaria, pemphigus vulgaris, bullous pemphigoid and cutaneous lupus erythematosus), hematological disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing disorders (such as, for example, inner ear disease and hearing loss), Behcet's syndrome, Raynaud's, organ transplantation, graft-versus-host disease (GVHD), and autoimmune endocrine disorders (such as, for example, diabetes-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)). More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

In some aspects, the subject has a proliferative disorder selected from non-Hodgkin's lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and marginal zone B-cell lymphoma (MZBL); and leukemias such as hairy cell leukemia (HCL), variant hairy cell leukemia (VHL-B), acute lymphoblastic leukemia (ALL) such as Philadelphia chromosome positive ALL (Ph+ALL) or Philadelphia chromosome negative ALL (Ph-ALL). [Fielding A, Haematologica. 2010 Jan; 95(1): 8-12].

In some aspects, the subject has diffuse large B-cell lymphoma.

Patient selection.

In some aspects, individuals are selected as suitable for treatment with combination therapy before treatment is administered.

In this context, individuals considered suitable for treatment are individuals expected to benefit from or respond to treatment. Individuals may have or be suspected of having cancer, or be at risk of developing cancer. Individuals may have been diagnosed with cancer. In particular, individuals may have or be suspected of having lymphoma, or may be at risk of developing lymphoma. In some cases, individuals may have or be suspected of having, or be at risk of developing, a solid cancer that has tumor-associated non-neoplastic cells expressing CD19, such as infiltrating cells expressing CD19.

In some aspects, individuals are selected based on the amount or pattern of CD19 expression. In some aspects, selection is based on cell surface expression of CD19.

In some aspects, the target is PI3K. In some aspects, the choice is based on PI3K expression.

In some respects, the choice is based on the levels of both cell surface CD19 and PI3K.

In some cases, expression of the target is determined in a specific tissue of interest. For example, in a sample of lymphoid tissue or tumor tissue. In some cases, expression of the target is determined systemically. For example, in a sample of circulating fluid such as blood, plasma, serum, or lymph.

In some aspects, an individual is selected as suitable for treatment due to the presence of target expression in a sample. In such cases, individuals without target expression may be considered ineligible for treatment.

In other aspects, the target expression level is used to select an individual that is suitable for

- 23 047973 for treatment. When the expression level of the target is above the threshold level, the individual is considered suitable for treatment.

In some aspects, the presence of CD19 and/or in cells in a sample indicates that the individual is suitable for treatment with a combination comprising an anti-CD19 ADC and a PI3K inhibitor or secondary agent. In other aspects, the amount of CD19 and/or expression must be above a threshold level to indicate that the individual is suitable for treatment. In some aspects, observing a change in CD19 and/or localization in a sample compared to a control indicates that the individual is suitable for treatment.

In some aspects, the individual is determined to be eligible for treatment if cells obtained from lymph nodes or extranodal sites react with antibodies to CD19 and/or as determined by IHC.

In some aspects, the patient is considered suitable for treatment if at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more of all cells in the sample express CD19. In some aspects described herein, the patient is considered suitable for treatment if at least 10% of the cells in the sample express CD19.

In some aspects, a patient is considered suitable for treatment if it is expressed in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of all cells in a sample. In some aspects described herein, a patient is considered suitable for treatment if it is expressed in at least 10% of the cells in a sample.

In some aspects, the individual is selected for treatment based on their current or previous treatment regimen. In some embodiments, the individual is selected for treatment with an anti-CD19 ADC if said individual has received treatment with a PI3K inhibitor or a secondary agent. In some embodiments, the individual is selected for treatment with an anti-CD19 ADC if said individual has received treatment with a PI3K inhibitor or a secondary agent. In some cases, the individual is selected for treatment if said individual is refractory to treatment (or further treatment) with a PI3K inhibitor or a secondary agent. In some cases, the PI3K inhibitor may be idelalisib or copanlisib. In some cases, the secondary agent may be bendamustine, bortezomib, lenalidomide, or olaparib. In those embodiments where the individual is receiving or has received treatment with a PI3K inhibitor or secondary agent, the anti-CD19 ADC can be administered in combination with the PI3K inhibitor or secondary agent, or without further administration of the PI3K inhibitor or secondary agent.

In some embodiments, the anti-CD19 ADC is administered to the selected individual in combination with a PI3K inhibitor or a secondary agent. In some embodiments, the anti-CD19 ADC is administered to the selected individual without continued administration of a PI3K inhibitor or a secondary agent.

Preferably, the PI3K inhibitor is idelalisib or copanlisib. Preferably, the secondary agent is olaparib or bendamustine.

The term refractory to treatment (or further treatment) with a PI3K inhibitor (or secondary agent) is used herein to mean that a disease (e.g., cancer) does not respond or ceases to respond to administration of a PI3K inhibitor or secondary agent given as monotherapy. In some embodiments, individuals with refractory NHL are identified using the response assessment criteria described in Cheson et al, 2014 (South Asian J Cancer. 2014 Jan-Mar; 3(1): 66-70). In this document, non-responders are defined as those who have either (i) an increase of >50% from the nadir of the sum of the products of the diameters of any previously identified abnormal nodule, or (ii) the appearance of any new lesion during or at the end of therapy. In some embodiments, individuals with refractory leukemia are identified as patients with stable or progressive disease who have completed one full cycle of treatment, or as patients who achieve a partial response after two or more full cycles of treatment.

Samples.

The sample may comprise or be obtained from: a quantity of blood; a quantity of serum obtained from the blood of an individual, which may include the liquid portion of the blood obtained after removal of the fibrin clot and blood cells; a quantity of pancreatic juice; a tissue sample or biopsy; or a cell isolated from said individual.

The sample may be taken from any tissue or bodily fluid. In certain aspects, the sample may include or be obtained from a tissue sample, biopsy, resection, or isolated cells from said individual.

In certain aspects, the sample is a tissue sample. The sample may be a tumor tissue sample, such as malignant tumor tissue. The sample may be obtained from a tumor biopsy. In some aspects, the sample is a lymphoid tissue sample, such as a lymphoid lesion sample or a lymph node biopsy. In some cases, the sample is a skin biopsy.

- 24 047973

In some aspects, the sample is taken from a bodily fluid, more preferably from a fluid circulating in the body. Accordingly, the sample may be a blood sample or a lymph sample. In some cases, the sample is a urine sample or a saliva sample.

In some cases, the sample is a blood sample or a sample obtained from blood. A sample obtained from blood may be a selected fraction of an individual's blood, such as a selected fraction containing cells or a fraction of plasma or serum.

The selected cell-containing fraction may comprise cell types of interest, which may include white blood cells (WBC), in particular peripheral blood mononuclear cells (PBMC) and/or granulocytes and/or red blood cells (RBC). Accordingly, the methods of the present invention may include detecting the first target polypeptide or nucleic acid in blood, in white blood cells, peripheral blood mononuclear cells, granulocytes and/or red blood cells.

The sample may be fresh or archival. For example, archival tissue may be obtained when a patient is initially diagnosed or biopsied at recurrence. In some aspects, the sample is a freshly obtained biopsy.

The first target polypeptide may be CD19.

Individual status.

An individual may be an animal, mammal, placental mammal, marsupial (e.g., kangaroo, wombat), monotreme (e.g., platypus), rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), lagomorph (e.g., rabbit), avian (e.g., bird), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), pig (e.g., swine), ovine (e.g., sheep), bovine (e.g., cow), primate, monkey (e.g., ape or monkey), ape (e.g., marmoset, baboon), great apes (e.g., gorilla, chimpanzee, orangutan, gibbon) or human.

Furthermore, the individual may be of any developmental form, such as a fetus. In one preferred embodiment, the individual is a human. The terms subject, patient, and individual are used interchangeably herein.

In some aspects described herein, the individual has or is suspected of having a risk of cancer or has been identified as being at risk of cancer. In some aspects described herein, the individual has already been diagnosed with cancer. An individual may be diagnosed with non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and marginal zone B-cell lymphoma (MZBL), and leukemias such as hairy cell leukemia (HCL), variant hairy cell leukemia (VHL-B), and acute lymphoblastic leukemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ALL) or Philadelphia chromosome-negative ALL (Ph-ALL). [Fielding A., Haematologica. 2010 Jan; 95(1): 8-12].

In some cases, the individual was diagnosed with non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphocytic lymphoma (CLL), and marginal zone B-cell lymphoma (MZBCL); and leukemias such as hairy cell leukemia (HCL), variant hairy cell leukemia (VHL-B), acute lymphoblastic leukemia (ALL) such as Philadelphia chromosome-positive ALL (Ph+ ALL) or Philadelphia chromosome-negative ALL (Ph- ALL). [Fielding A, Haematologica. 2010 Jan; 95(1): 8-12].

In some cases, the individual has already been diagnosed with a solid cancer containing infiltrating cells expressing CD19+.

The individual may be receiving or has received therapeutic treatment for this cancer. The subject may or may not have previously received ADCX19. In some cases, the cancer is a lymphoma, including non-Hodgkin lymphoma.

The individual may be receiving or has been treated with a PI3K inhibitor or a secondary agent. In some cases, the individual may be refractory to treatment (or further treatment) with a PI3K inhibitor or a secondary agent. In some cases, the PI3K inhibitor may be idelalisib or copanlisib. In some cases, the secondary agent may be bendamustine, bortezomib, lenalidomide, or olaparib. In embodiments where the individual is receiving or has been treated with a PI3K inhibitor or a secondary agent, the anti-CD19 ADC may be administered in combination with the PI3K inhibitor or secondary agent without continued administration of the PI3K inhibitor or secondary agent.

- 25 047973

Controls.

In some aspects, target expression in an individual is compared to target expression in a control. Controls are useful for confirming the accuracy of staining and identifying experimental artifacts.

In some cases, the control may be a reference sample or a reference data set. A reference may be a sample that was previously obtained from a person with a known degree of fitness. A reference may be a data set obtained from the analysis of a reference sample.

Controls can be positive controls in which the target molecule is known to be present or expressed at high levels, or negative controls in which the target molecule is known to be absent or expressed at low levels.

Controls may be tissue samples taken from individuals known to benefit from the treatment. The tissue may be the same type as the sample being tested. For example, a tumor tissue sample from an individual may be compared to a control tumor tissue sample from an individual known to be suitable for the treatment, such as an individual who has previously responded to treatment.

In some cases, the control may be a sample obtained from the same person as the test sample, but from tissue known to be healthy. In this way, a cancerous tissue sample from an individual can be compared to a non-cancerous tissue sample.

In some cases, the control is a cell culture sample.

In some cases, the test sample is analyzed prior to incubation with the antibody to determine the level of background staining inherent in the sample.

In some cases, an isotype control is used. Isotype controls use antibodies of the same class as the specific target antibodies, but they do not react with the sample. Such controls are useful for distinguishing non-specific interactions with the target specific antibody.

Such methods may include interpretation of morphology and immunohistochemistry by a hematopathologist to ensure accurate interpretation of the test results. The method may include confirming that the expression profile correlates with the expected profile. For example, when analyzing the expression level of CD19 and/or PI3K, the method may include confirming that the expression is observed as membrane staining with a cytoplasmic component in the test sample. The method may include confirming that the target signal to noise ratio is above a threshold level, allowing specific and non-specific background signals to be clearly distinguished.

Treatment methods.

The term treatment, as used herein in the context of treating a pathological condition, generally refers to treatment and therapy, whether in humans or animals (e.g., in veterinary medicine), that achieves some desired therapeutic effect, such as inhibiting the progression of a pathological condition and includes reducing the rate of progression, halting the rate of progression, regressing a pathological condition, ameliorating a pathological condition, and curing a pathological condition. The term also includes treatment as a prophylactic measure (i.e., prophylaxis, prevention).

As used herein, the term "therapeutically effective amount" or "effective amount" refers to that amount of an active compound or a material, composition, or dose comprising the active compound that is effective to produce some desired therapeutic effect commensurate with a reasonable benefit/risk ratio when administered in accordance with the desired treatment regimen.

Similarly, the term prophylactically effective amount refers to that amount of the active compound or material, composition or dose comprising the active compound that is effective to produce some desired prophylactic effect commensurate with a reasonable benefit/risk ratio when administered in accordance with the desired treatment regimen.

The present document describes methods of treatment. Also provided is a method of treatment comprising administering to a subject in need of such treatment a therapeutically effective amount of an anti-CD19 ADC and a PI3K inhibitor or a secondary agent. The term therapeutically effective amount means an amount sufficient to demonstrate a benefit to the subject. Such benefit may include at least an alleviation of at least one symptom. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the pathological condition being treated. The administration of treatment, such as dose decisions, is the responsibility of general practitioners and other physicians. The subject may have been assessed to determine whether he or she meets the criteria for receiving treatment according to the methods described herein. The method of treatment may include the step of determining whether the subject is eligible to receive treatment using the method described herein.

Ahtu-CD19 ADC contains an antibody to CD19. The antibody to CD19 may be an antibody

- 26 047973

RB4v1.2. The ADC may comprise a drug that is a PBD dimer. The ADC may be ADCx19. The ADC may be an ADC as described in WO2014/057117.

The PI3K inhibitor may be idelalisib, copanlisib, duvelisib, taselisib, buparlisib, alpelisib, umbralisib, dactolisib and voxtalisib. Preferably, the PI3K inhibitor is idelalisib or copanlisib.

The secondary agent may be:

(a) Bendamustine;

(b) Lenalidomide;

(c) a proteasome inhibitor such as bortezomib, carfilzomib, ixazomib, oprozomib, and salinosporamide A; or (d) a PARP inhibitor such as olaparib, CEP-9722, BMN-673/talazoparib, rucaparib, iniparib/SAR24-550/BSI-201, veliparib (ABT-888), niraparib/MK-4827, BGB-290, 3-aminobenzamide, and E7016.

Treatment may include administration of an αanti-CD19 ADC/PI3K inhibitor combination or a secondary agent alone or in additional combination with other treatments, either concomitantly or sequentially, depending on the condition being treated.

A typical treatment regimen with a combination of anti-CD19 ADC plus PI3K inhibitor involves the following steps:

(1) identification of an individual who has been or is being treated with a PI3K inhibitor such as idelalisib or copanlisib;

(2) administering to the individual an anti-CD19 ADC, such as ADCx19; and, optionally (3) administering to the individual a PI3K inhibitor, such as idelalisib, in combination with an anti-CD19 ADC (e.g., concurrently with the ADC or subsequent to the ADC).

A typical treatment regimen with a combination of anti-CD19 ADC plus a secondary agent includes the following steps:

(1) identification of an individual who has been or is being treated with a secondary agent such as bendamustine, bortezomib, lenalidomide, or olaparib;

(2) administering to the individual an anti-CD19 ADC, such as ADCx19; and, optionally (3) administering to the patient a secondary agent, such as bendamustine, bortezomib, lenalidomide, or olaparib, in combination with an anti-CD19 ADC (e.g., concomitantly with the ADC or subsequent to the ADC).

Examples of treatment and therapy include, but are not limited to, chemotherapy (administration of active agents, including, for example, drugs such as chemotherapeutic agents); surgery; and radiation therapy.

A chemotherapeutic agent is a chemical compound used in the treatment of cancer, regardless of the mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, plant spindle alkaloids, cytotoxic/antineoplastic antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in targeted therapy and conventional chemotherapy.

Examples of chemotherapeutic agents include: lenalidomide (REVLIMID®, Celgene), vorinostat (ZOLINZA®, Merck), panobinostat (FARYDAK®, Novartis), mocetinostat (MGCD0103), everolimus (ZORTRESS®, CERTICAN®, Novartis), bendamustine (TREAKISYM®, RIBOMUSTIN®, LEVACT®, TREANDA®, Mundipharma International), erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen (^)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®) and doxorubicin (ADRIAMYCIN®), Akti1/2, Htamoxifen PPD and rapamycin.

Other examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006,

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Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (without cremophor), paclitaxel albumin nanoformulations (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); Alkyl sulfonates such as busulfan, improsulfan, and piposulfan; Aziridines such as benzodopa, carboquone, meturedopa, and uredopa; Ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamin; Acetogenins (especially bullatacin and bullatacinone); Camptothecin (including the synthetic analogue topotecan); Bryostatin; Callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); Cryptophycins (especially cryptophycin 1 and cryptophycin 8); Dolastatin; Duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); Eleutherobin; Pancratistatin; Sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, calicheamicin gamma-P, calicheamicin omega-P (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates such as clodronate; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-Bnorleucine, morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, kelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; Androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Adrenal suppressants such as aminoglutethimide, mitotane, trilostane; Folic acid replenisher such as frolinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; demecolcine; diaziquone; elphornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); azoxan; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anhydrine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside (Ara-C); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum-based analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above. Combinations of agents such as CHP (doxorubicin, prednisone, cyclophosphamide) or CHOP (doxorubicin, prednisone, cyclophosphamide, vincristine) may be used.

Also included within the definition of chemotherapeutic agent are: (i) antihormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestane, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis) and ARIMIDEX® (anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue); (iv) protein kinase inhibitors, such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, especially those that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as PKC-alpha, Raf and H-Ras, such as oblimersen (GEN-28 047973

ASENSE®, Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, e.g., ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors, such as LURTOTECAN®; ABARELIX® rmRH; (ix) antiangiogenic agents, such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

Also included in the definition of chemotherapeutic agent are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), pertuzumab (PERJETA™, OMNITARG™, 2C4, Genentech), trastuzumab (FJERCEPTIN®, Genentech), MDX-060 (Medarex), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates described herein include: alemtuzumab, apolizumab, azelizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motivizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, omalizumab, palivizumab, pascolizumab, pekfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, recivizumab, rovelizumab, ruplizumab, sibrotuzumab, sontuzumab, takatuzumab, tetraxetane, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleuquin, tucusituzumab, umavizumab, urtoxazumab and visilizumab.

The compositions of the present invention are preferably pharmaceutical compositions. The pharmaceutical compositions according to the invention and for use according to the invention may contain, in addition to the active ingredient, i.e. the conjugated compound, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact characteristics of the carrier or other material will depend on the route of administration, which may be oral or injection, such as cutaneous, subcutaneous or intravenous injection.

Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powder or liquid. A tablet may contain a solid carrier or adjuvant. Liquid pharmaceutical compositions typically contain a liquid carrier such as water, petrolatum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may contain a solid carrier such as gelatin.

For intravenous, cutaneous or subcutaneous injection or injection into the site of injury, the active ingredient will be in the form of a parenterally acceptable aqueous solution, free of pyrogens and having a suitable pH, isotonicity and stability. Those skilled in the art can successfully prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride for injection, Ringer's solution for injection, Ringer's lactate for injection. If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives can be included.

Dose.

It will be appreciated by one skilled in the art that suitable doses of the anti-CD19 ADC and/or PI3K inhibitor or secondary agent and compositions containing these active elements may vary from subject to subject. Determination of the optimal dose typically involves the principle of balancing the level of therapeutic effect with any risk or deleterious side effects. The selected dose level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of elimination of the compound, the duration of treatment and the presence of other drugs, compounds and/or materials used in combination, the severity of the pathological condition and the species, sex, age, weight, pathological condition, general health and previous medical history of the subject. The amount of compound and the route of administration will ultimately be determined by a physician, veterinarian or clinician, although typically the dose is selected to achieve local concentrations at the site of action that result in the desired effect without significant detrimental effects or undesirable side effects.

In some aspects, the dose of the anti-CD19 ADC is determined by the expression of CD19 observed in a sample obtained from the subject. Thus, the level or location of CD19 expression in the sample may indicate that a higher or lower dose of the anti-CD19 ADC is required. For example, a high level of CD19 expression may indicate that a higher dose of the anti-CD19 ADC is appropriate.

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In some cases, high CD19 expression may indicate the need to administer another agent in addition to the anti-CD19 ADC. For example, administering an anti-CD19 ADC along with a chemotherapeutic agent. High CD19 expression may indicate more aggressive therapy.

In some aspects, the dose of the PI3K inhibitor or secondary agent is determined by the expression observed in a sample obtained from the subject. Thus, the level or location of expression in the sample may indicate that a higher or lower dose of the PI3K inhibitor or secondary agent is needed. For example, a high level of PI3K expression may indicate that a higher dose of the PI3K inhibitor or secondary agent would be appropriate. In some cases, a high level of PI3K expression may indicate the need to administer another agent in addition to the PI3K inhibitor or secondary agent. For example, administering a PI3K inhibitor or secondary agent in combination with a chemotherapeutic agent. A high level of PI3K expression may indicate more aggressive therapy.

Administration may be performed in a single dose, continuously, or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods for determining the most effective agents and doses are well known to those skilled in the art and will vary depending on the formulation used for therapy, the purpose of therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations may be performed with the choice of dose level and schedule by the attending physician, veterinarian, or clinician.

Typically, a suitable dose of each active compound is in the range of about 100 ng to about 25 mg (more typically about 1 mcg to about 10 mg) per kilogram of subject body weight per day. When the active compound is a salt, ester, amide, prodrug or similar substance, the amount administered is calculated on the basis of the parent compound, and therefore the actual weight to be taken into account is increased proportionally.

In one embodiment, each active compound is administered to a human subject according to the following dosing regimen: about 100 mg 3 times daily.

In one embodiment, each active compound is administered to a human subject according to the following dosing regimen: about 150 mg twice daily.

In one embodiment, each active compound is administered to a human subject according to the following dosing regimen: about 200 mg twice daily.

However, in one embodiment, each conjugate compound is administered to a human subject according to the following dosing regimen: about 50 or about 75 mg, 3 or 4 times per day.

In one embodiment, each conjugate compound is administered to a human subject according to the following dosing regimen: about 100 or about 125 mg, 2 times per day.

For anti-CD19 ADCs where the ADC carries a PBD, the dosage amounts described above may apply to the conjugate (including the PBD moiety and linker to the antibody) or to an effective amount of the proposed PBD compound, such as the amount of compound that is released upon cleavage of the linker.

The Ahtu-CD19 ADC comprises an antibody to CD19. The antibody to CD19 may be the RB4v1.2 antibody. The ADC may comprise a drug that is a PBD dimer. The anti-CD19 ADC may be an ADCx19. The ADC may be an ADC as described in WO2014/057117.

The PI3K inhibitor may be idelalisib, copanlisib, duvelisib, taselisib, buparlisib, alpelisib, umbralisib, dactolisib and voxtalisib. Preferably, the PI3K inhibitor is idelalisib or copanlisib.

The secondary agent may be:

(a) Bendamustine;

(b) Lenalidomide;

(c) a proteasome inhibitor such as bortezomib, carfilzomib, ixazomib, oprozomib, and salinosporamide A; or (d) a PARP inhibitor such as olaparib, CEP-9722, BMN-673/talazoparib, rucaparib, iniparib/SAR24-550/BSI-201, veliparib (ABT-888), niraparib/MK-4827, BGB-290, 3-aminobenzamide, and E7016.

Antibodies.

The term antibody is used in the broadest sense herein and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), intact antibodies (also described as full-length antibodies), and antibody fragments, as long as they exhibit the desired biological activity, such as the ability to bind CD19 (Miller et al (2003) Jour, of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or from other species such as rabbit, goat, sheep, horse, or camel.

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An antibody is a protein produced by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). The target antigen typically has multiple binding sites, also called epitopes, recognized by complementarity determining regions (CDRs) on multiple antibodies. Each antibody, which binds specifically to a different epitope, has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody may comprise a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds an antigen of a target of interest or a portion thereof, wherein such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), subclass, or allotype (e.g., G1m1, G1m2, G1m3, non-G1m1 [i.e., any allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2, and Km3) of a human immunoglobulin molecule. Immunoglobulins can come from any species, including human, mouse, or rabbit origin.

Antibody fragments comprise a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementarity determining region) and epitope-binding fragments of any of the foregoing that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

As used herein, the term monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by the hybridoma method first described in Kohler et al (1975) Nature 256:495, or can be produced by recombinant DNA techniques (see US 4,816,567). Monoclonal antibodies can also be isolated from antibody phage libraries using the methods described in, for example, Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol, 222: 581-597, or from transgenic mice carrying the entire human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).

Monoclonal antibodies as used herein specifically include chimeric antibodies in which a portion of the heavy and/or light chain is identical to or homologous with corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass and the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies derived from another species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see US 4,816,567; and Morrison et al (1984) Proc. Natl. Acad Sci. USA, 81:68516855). Chimeric antibodies include primatized antibodies that contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys or apes) and human constant region sequences.

An intact antibody as used herein is an antibody comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., native sequence human constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more effector functions that relate to the biological activity that is attributed to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent

- 31 047973 cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and inhibition of cell surface receptors such as the B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be classified into different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the heavy chain that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known.

Brief description of graphic materials

Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying figures, in which:

Fig. 1. Sequences.

The disclosure includes a combination of the described aspects and preferred features, except where such a combination is clearly impermissible or clearly not permitted.

The section headings used in this description are for organizational purposes only and should not be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated by way of example with reference to the accompanying figures. Additional aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Throughout this specification, including the appended claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" shall be understood as implying the inclusion of the stated integer or step or group of integers or steps, but not the exclusion of any other integer, step or group of integers or steps.

It should be noted that, as used in the description and the appended claims, the singular forms include plural references unless the context clearly dictates otherwise. As used herein, ranges may be expressed as from about one specific value and/or to about another specific value. When such a range is expressed, another embodiment includes from one specific value and/or to another specific value. Similarly, when values are expressed as approximations, using the term about, it should be understood that the specific value forms another embodiment.

Preferred embodiments

The following paragraphs describe some specific preferred embodiments of the present invention:

1. A method of treating cancer in an individual, comprising administering to the individual an effective amount of an anti-CD19 ADC and a PI3K inhibitor;

and the said anti-CD19 ADC has the chemical structure:

where Ab is an antibody that binds to CD19.

2. The method according to claim 1, characterized in that the anti-CD19 ADC comprises an antibody having:

(i) a VH domain having a sequence according to any of SEQ ID NO. 1, 2, 3, 4, 5 or 6, and a VL domain having a sequence according to any of SEQ ID NO. 7, 8, 9, 10, 11 or 12;

(ii) a VH domain having the sequence according to SEQ ID NO. 1 and a VL domain having the sequence according to SEQ ID NO. 7;

(iii) a VH domain having the sequence according to SEQ ID NO. 2 and a VL domain having the sequence according to SEQ ID NO. 8;

- 32 047973 (iv) a VH domain having a sequence according to SEQ ID NO. 3 and a VL domain having a sequence according to SEQ ID NO. 9;

(v) a VH domain having the sequence according to SEQ ID NO. 4 and a VL domain having the sequence according to SEQ ID NO. 10;

(vi) a VH domain having the sequence according to SEQ ID NO. 5 and a VL domain having the sequence according to SEQ ID NO. 11; or (vii) a VH domain having the sequence according to SEQ ID NO. 6 and a VL domain having the sequence according to SEQ ID NO. 12.

3. The method according to claim 1 or 2, characterized in that the anti-CD19 ADC is ADCT-402.

4. The method according to any one of claims 1-3, wherein the treatment comprises administering the anti-CD19 ADC before the PI3K inhibitor or simultaneously with the PI3K inhibitor, or after the PI3K inhibitor.

5. A method according to any of the preceding paragraphs, characterized in that the individual:

(i) has cancer or has been determined to have cancer;

(ii) has or has been determined to have a cancer that expresses CD19 or has tumor-associated CD19+ non-neoplastic cells, such as infiltrating CD19+ cells;

(iii) is receiving treatment with a PI3K inhibitor or;

(iv) has received treatment with a PI3K inhibitor; and/or (v) is refractory to treatment or further treatment with a PI3K inhibitor;

6. The method according to any of the preceding paragraphs, characterized in that the cancer:

(i) is selected from the group consisting of: non-Hodgkin's lymphoma, including diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and marginal zone B-cell lymphoma (MZBL), and leukemias such as hairy cell leukemia (HCL), variant hairy cell leukemia (VHL-B), and acute lymphoblastic leukemia (ALL), such as Philadelphia chromosome positive ALL (Ph+ALL) or Philadelphia chromosome negative ALL (PhALL); or (ii) is diffuse large B-cell lymphoma (DLBCL); optionally, the DLBCL is relapsed or refractory.

7. The method according to any of the preceding claims, wherein the PI3K inhibitor is idelalisib, copanlisib, duvelisib, tazelisib, buparlisib, alpelisib, umbralisib, dactolisib or voxtalisib.

8. The method of claim 7, wherein the PI3K inhibitor is idelalisib.

9. The method of claim 7, wherein the PI3K inhibitor is copanlisib.

Examples

In the following examples:

Cell lines expressing CD19 suitable for use in the examples include Ramos, Daudi, Raji, WSU-DLCL, and NALM-6 cells.

Disease A - Diffuse large B-cell lymphoma/DLBCL is an aggressive type of non-Hodgkin lymphoma that develops from B cells of the lymphatic system. It is the largest subgroup of non-Hodgkin lymphomas.

Mantle cell lymphoma (MCL) is a rare B-cell NHL that most often affects men over 60 years of age. The disease can be aggressive (fast-growing), but in some patients it can also be more indolent (slow-growing). MCL accounts for about five percent of all NHL.

Disease C - Follicular lymphoma/FL is a fairly indolent type of NHL with a long survival time but is very difficult to cure; it can also transform into more aggressive forms of lymphoma.

Example 1.

In a separate experiment, CD19-expressing cell lines were incubated for 0, 6, 24, and 48 h with etoposide (negative control) and oxaliplatin (positive control), 1 μg/ml anti-CD19 ADC (ADC targeting CD19 with a PBD dimer as the active payload), 1 μg/ml anti-CD19 antibody (antibody in the ADC), and 1 μg/ml B12-SG3249 (a non-binding control ADC with the same PBD payload as the anti-CD19 ADC).

Following incubation, cells were washed and fed with human dendritic cells (DCs) for an additional 24 h. DC activation was subsequently measured by increased surface expression of CD86 in the DC population (as determined by flow cytometry) and by measuring DC-mediated release of IL-8 and MIP2.

Example 2.

The aim of this study is to preliminarily evaluate the safety, tolerability, pharmacological and clinical activity of this combination.

The following cancer types were selected for the study: Disease A, Disease B, and Disease C.

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Evidence of efficacy as single agents exists for both drugs:

anti^19 ADC (see, for example, WO2014/057117, WO2016/166298, WO2014/057122 and WO2016/166307).

PI3K inhibitor or secondary agent (see KS Peggs et al, 2009, Clinical and Experimental Immunology, 157: 9–19 [doi: 10.1111/j.l365-2249.2009.03912.x]).

The primary objective of this study is to investigate whether these agents can be safely combined and, if so, to identify dose(s) and regimens appropriate for further investigation. The study will also evaluate whether each combination produces pharmacologic changes in tumors that suggest potential clinical benefit.

Additionally, the study will provide preliminary evidence that the combination may increase the rate and duration of responses compared with published data for treatment with anti-CD19 ADC or PI3K inhibitor monotherapy or a secondary agent.

Each disease group may include a subset of patients previously treated with a PI3K inhibitor or a secondary agent to determine whether combination therapy can overcome resistance to PI3K inhibitor or secondary agent therapy. Specific molecular selection is not intended for each disease, as currently available data generally do not support exclusion of patients based on validated molecular diagnostic tests.

Rationale for starting dose of anti-CD19 ADC.

The RDE for the established ADC (in mcg/kg administered every three weeks) will be used for all patients in this study. To ensure patient safety, a starting dose below the RDE will be used; the starting dose level will be one at which patient benefit can still be demonstrated in the ADC1 study, suggesting that patients enrolled at this dose level will receive at least some benefit from participation.

Rationale for initial dose of PI3K inhibitor or secondary agent.

The RDE for the established PI3K inhibitor or secondary agent (in mcg/kg administered every three weeks) will be used for all patients in this study. To ensure patient safety, a starting dose below the RDE will be used; the starting dose level will be one at which patient benefit can still be demonstrated in the SA1 study, suggesting that patients enrolled at this dose level will receive at least some benefit from participation.

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Objectives and related endpoints.

Object End point Primary objective To characterize the safety and tolerability of ADCs in combination with a PI3K inhibitor or secondary agent and to identify recommended doses and regimens for future studies. To determine the frequency and severity of treatment-emergent AEs and SAEs To determine changes between baseline and post-baseline laboratory parameters and vital signs To determine the incidence of dose-limiting toxicities (DLTs) during the first treatment cycle (dose escalation only) To determine the incidence of dose interruptions and dose reductions Secondary objectives To evaluate the clinical activity of the combination of an ADC with a PI3K inhibitor or secondary agent To characterize the pharmacokinetic (PK) profile of each of the two compounds, an ADC and a PI3K inhibitor or secondary agent To test for evidence of immunogenicity and ADA to the ADC ORR (objective response rate), DOR (duration of response), PFS (progression-free survival), OS (overall survival) AUC and Cmax for each compound Check anti-drug antibodies (ADA) before, during and after ADC treatment Research objectives To investigate the potential correlation of PK profiles with safety/tolerability and efficacy. Determine correlation coefficients between AUC and/or Cmax for each compound or compound measure and any safety variables To characterize changes in immune infiltrate in tumors. To characterize changes in levels of circulating cytokines in plasma and activation markers in circulating immune cells. or efficacy Perform immunohistochemical analysis of tumor biopsies before and after treatment, Perform measurements (e.g., ELISA) of immunologically relevant cytokines in plasma or serum; determine staining levels for markers of circulating immune cell activation (e.g., FACS)

Study design.

This study is a multicenter, open-label, phase Ib study to characterize the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of ADC in combination with a PI3K inhibitor or secondary agent in patients with disease A, disease B, and disease C.

The study consists of a dose escalation portion followed by a portion using the previously established maximum tolerated dose in an expanded population.

Dose escalation begins with reduced starting doses (compared to their respective recommended phase 2 or licensed dose levels) for both the ADC and the PI3K inhibitor or secondary agent to ensure patient safety. Starting doses will be

- 35 047973 33% (or 50%) of the RDE for each compound. Thereafter, the doses of the PI3K inhibitor or secondary agent will be initially escalated until the RDE or licensed dose is reached, or, if necessary, a lower dose for tolerability reasons. The dose of the ADC will then be escalated until the RDE for the combination treatment is reached. This is shown in Fig. 1:

The estimated safe starting dose is 33% of the estimated effective dose for both compounds, but may need to be adjusted to a lower or higher dose depending on the individual risk profile for the combination. Compound 1 should be

a compound for which an effective clinical dose (at 100%) has been established and is therefore expected to be rapidly delivered to trial patients by increasing the dose of that compound first.

If a dose combination is determined to be safe, it may be tested in additional patients to confirm safety and tolerability at that dose level. Further dose adjustments may be made for each compound and/or the regimen may be modified.

Dose escalation of the combination will be guided by a Bayesian logistic regression model (BLRM) based on any dose-limiting toxicities (DLTs) observed in the first (or first two, TL) cycles of therapy. The use of BLRM is a well-established method for estimating the maximum tolerated dose (MTD)/recommended dose for escalation (RDE) in cancer patients. The adaptive BLRM will be guided by the Escalation with Overdose Control (EWOC) principle to manage the risk of DLTs in future patients enrolled in the study. The use of adaptive Bayesian response models for small datasets has been approved by the FDA and EMEA (Small Group Clinical Trials Guidelines, February 1, 2007) and is supported by numerous publications (Babb et al. 1998, Neuenschwander et al. 2008).

Decisions on new dose combinations are made by the investigators and study staff on the sponsor's side in the dose escalation safety signal (DESC) based on an analysis of patient tolerability and safety information (including a summary of the BLRM DLT risk, if applicable) together with PK, PD, and preliminary information available at the time of decision making.

Once the MTD/RDE for the combination has been determined, an extension portion of the study can be initiated to further evaluate safety, tolerability, and preliminary efficacy.

For combinations with 10, changes in immune infiltrate in tumors will also be characterized after combination treatment according to target disease indications.

Given the available prior clinical experience with the agents in this study, it is expected that in most cases the combination dose can be determined without testing a large number of dose levels or schedules. To assess the pharmacodynamic activity of the combinations, patients will be asked to undergo tumor biopsy at baseline and again after approximately two cycles of therapy.

For combinations 10: The degree of change in tumour infiltration of immune cells, including lymphocytes and macrophages, will be taken into account when deciding on any potential benefit.

Part of the dose increase.

During the dose-escalation portion of the study, patients will be treated with a fixed dose of ADC administered intravenously and escalating doses of a PI3K inhibitor or secondary

- 36,047,973 agents until the RDE for the PI3K inhibitor or secondary agent is reached. Subsequently, the ADC doses are increased (across groups) while the dose of the PI3K inhibitor or secondary agent remains constant.

Between two and approximately 3-4 patients with disease A, disease B, or disease C will be treated in each dose-escalating cohort until the MTD/RDE is determined.

There will be a 24-hour observation period before the second patient is enrolled in the Dose Level 1 cohort. The DLT observation period at each dose level is either 1 cycle (3 weeks) or 2 cycles (6 weeks), as prescribed by the respective regulators for therapy 10, after which a determination will be made whether to escalate to the next dose level, remain at the current dose level, or decrease to the previous dose level for the next cohort. There will be no reduction from Dose Level 1. Dose escalation is not permitted in the same patient.

Dose escalation is not permitted if 2 or more patients do not have complete EBRT data during the first cycle at any given dose level. Dose escalation will be guided by the mCRM with a target EBRT rate of 30% and an equivalence interval of 20% to 35%, and dose escalation with overdose monitoring (EWOC) and no missed doses.

Patients will be allocated to a cohort that is actively recruiting. Dose escalation will be performed in each combination after completion of one treatment cycle. Safety assessments, including adverse events (AEs) and laboratory parameters, will be closely monitored for all enrolled patients to identify any EBRTs. A single MTD/RDE ratio will be defined; disease-specific MTD/RDE is not established.

The mCRM will be implemented for DE (dose escalation) under the oversight of a Dose Escalation Steering Committee (DESC). The DESC will approve each escalating dose level after reviewing all available safety data. PK data from patients at that dose level and previous dose levels may also be used to make the decision. The DESC may withhold dose escalation until the MTD is determined based on emerging PK, PD, toxicity, or response data.

Additional patients may be included at any dose level for further evaluation of safety and tolerability if at least 1 patient in the study achieved a partial response or better, or if DESC deems further evaluation of PK or PD data necessary to determine the RDE.

Dose escalation will be stopped after 3 groups (or at least 6 patients) have been consecutively assigned to the same dose level. If the MTD is not achieved, the recommended dose extension (RDE) will be determined. Before the MTD/RDE can be determined, at least 6 patients must be treated with the combination.

It is anticipated that paired tumor biopsies will be collected from patients during dose escalation. Analysis of these biopsies will contribute to a better understanding of the relationship between dose and pharmacodynamic activity of the combination.

Safety oversight by the DESC Dose Escalation Steering Committee, comprised of ADC Therapeutics and the investigators, will continually review patient safety during DE to determine whether the dose escalation schedule prescribed by the mCRM justifies changes. In addition to safety observations, PK and/or PD data may also be used to guide decisions. Intermediate doses may be prescribed following agreement between ADC Therapeutics and the investigators. DESC may continue to provide oversight during Part 2. A formal Data Safety Monitoring Board (DSMB) will not be used.

Part of the use of a previously established maximum tolerated dose in an expanded population.

Once the MTD/RDE is announced, the use-up portion of the previously established maximum tolerated dose in the expanded population may be initiated. The primary purpose of the use-up portion of the previously established maximum tolerated dose in the expanded population is to further evaluate the safety and tolerability of the study treatment in the MTD/RDE and to obtain a preliminary understanding of the efficacy of the combination compared to historical single agent efficacy data.

An important aim of the study is to assess changes in the level of tumour immune infiltrate in response to treatment. This will be assessed in paired tumour biopsies collected from patients, with a minimum of ten biopsy pairs analysed (biopsy samples must contain sufficient tumour for analysis) from patients treated in the MTD/RDE. If this is not possible, collection of these biopsies may be stopped. It is planned that at least 10 to 20 patients will be treated in each study arm.

Several different study arms will be opened, one for each disease. A total of nine study arms can be launched using the previously established maximum tolerated dose in the expanded population. If recruitment to any of these arms is not possible, then recruitment to that arm can be closed before the target of 10 to 20 patients is reached.

Each treatment group will be allowed to treat a maximum of about six patients who were treated and who had disease progression in the previous one.

- 37,047,973 single doses (i.e. not in combination) of PI3K inhibitor or secondary agent therapy. This number may be increased if the combination is promising to overcome resistance to the previous treatment with a single dose of PI3K inhibitor or secondary agent.

Patient population.

A study will be conducted in adult patients with advanced disease A, disease B, or disease C as described above. The investigator or designee must ensure that study treatment is offered only to patients who meet all of the following inclusion criteria and none of the exclusion criteria.

Inclusion criteria.

Patients eligible for inclusion in this study must meet all of the following criteria:

1. Written informed consent must be obtained before any procedures are commenced.

2. Age 18 years.

3. Patients with advanced/metastatic disease, with measurable disease as defined in RECIST version 1.1, whose disease is progressing despite standard therapy, or who are intolerant of standard therapy, or for whom standard therapy is not available. Patients must be in one of the following groups:

Disease A.

Disease B.

Disease C.

4. General condition index according to the ECOG scale 0-1 (or 2 TVS).

5. TVS: The patient must have a biopsyable lesion and be a candidate for tumor biopsy according to institutional guidelines. The patient must be willing to undergo a new tumor biopsy at baseline and again during therapy in this study.

6. Pre-treatment with a PI3K inhibitor or secondary agent or related compounds (i.e., the same MOA) is allowed.

Non-inclusion criterion.

Patients eligible for this study must not meet any of the following criteria:

1. History of severe hypersensitivity reactions to other mAbs (or to mAbs with the same backbone as the ADC, or to the same IO mAb, if applicable).

2. History of positive human serum ADA to the mAb backbone as in ADC.

3. Central nervous system (CNS) disease only (if applicable).

4. Symptomatic CNS metastases or evidence of leptomeningeal disease (MRI of the brain or previously documented cerebrospinal fluid (CSF) cytology).

Previously treated asymptomatic CNS metastases are permitted provided that the most recent treatment (systemic antineoplastic therapy and/or local radiotherapy) was completed >= 8 weeks prior to ^day 1 of dosing, except that low-dose steroids may be used in addition). Patients with discrete dural metastases are eligible.

5. A patient who has laboratory values outside the acceptable range, defined as:

Serum creatinine <= 1.5 x ULN (upper limit of normal). If serum creatinine is > 1.5, creatinine clearance (calculated using the Cockcroft-Gault formula or measured) must be > 60 mL/min/1.73 ^m2 for the patient to be eligible for study participation.

Total bilirubin level > 1.5 x ULN, except for patients with Gilbert's syndrome, who are excluded if total bilirubin level > 3.0 x ULN or direct bilirubin level > 1.5 x ULN.

Alanine aminotransferase (ALT) level > 3 x ULN, except for patients with liver tumor lesions, who are excluded if ALT level > 5 x ULN.

Aspartate aminotransferase (AST) level > 3 x ULN, except for patients with liver tumor lesions, who are excluded if AST level > 5 x ULN.

Absolute neutrophil count < 1.0 x 10e9/L.

Platelet count < 75 x 10e9/L.

Hemoglobin (Hgb) level < 8 g/dL.

Potassium, magnesium, calcium, or phosphate abnormality > CTCAE grade 1 despite appropriate replacement therapy.

6. Impaired cardiac function or clinically significant cardiac disease, including any of the following:

Clinically significant and/or uncontrolled cardiac disease such as congestive heart failure requiring treatment (NYHA stage III or IV) or uncontrolled hypertension,

- 38 047973 defined by systolic blood pressure (SBP) 160 mmHg and/or diastolic blood pressure (DBP) 100 mmHg with or without antihypertensive drugs.

QTcF> 470 ms for women or > 450 ms for men on screening ECG with Fridericia correction, congenital long QT syndrome.

Acute myocardial infarction or unstable angina < 3 months (months before study inclusion).

Clinically significant valvular heart disease with documented impairment of cardiac function.

Symptomatic pericarditis.

History of or current documented cardiomyopathy.

Left ventricular ejection fraction (LVEF) < 40% as determined by echocardiogram (ECHO) or radionuclide ventriculography (MUGA).

History or current presence of any clinically significant cardiac arrhythmia, such as ventricular, supraventricular, junctional or conduction disturbances (TVS qualifier: ... requiring a pacemaker or not controlled by medication).

Presence of unstable atrial fibrillation (ventricular response rate > 100 beats per minute).

Note: Patients with stable atrial fibrillation may be included in the study provided they do not meet other cardiac exclusion criteria.

Complete left bundle branch block (RBBB), bifascicular block.

Any clinically significant ST segment and/or T wave abnormalities.

7. Toxicity related to previous IO therapy that led to discontinuation of therapy. Adequately treated patients with drug-induced skin rash or with replacement therapy for endocrinopathies are not excluded, provided that these toxic effects did not lead to discontinuation of previous treatment.

8. Patients with an active, known or suspected autoimmune disease. Subjects with vitiligo, type 1 diabetes mellitus, residual hypothyroidism due to an autoimmune condition requiring only hormone replacement therapy, psoriasis not requiring systemic treatment, or conditions not expected to recur in the absence of an external trigger are permitted for inclusion provided the trigger can be avoided.

9. Human immunodeficiency virus (HIV) or active hepatitis B virus (HBV) or hepatitis C virus (HCV) infection.

Testing is not mandatory for participation. HCV testing should be considered if the patient is at risk for undiagnosed HCV (eg, history of injection drug use).

10. Malignancies not relevant to study treatment. Exceptions to this exclusion include the following: malignancies that were treated to cure and had not recurred within 2 years prior to study treatment; completely resected basal cell and squamous cell skin cancers; any malignancy considered painless and never requiring treatment; and completely resected carcinoma in situ of any type.

11. Systemic anticancer therapy within 2 weeks of the first dose of study drug. For cytotoxic agents that have significant delayed toxicity, such as mitomycin C and nitrosoureas, the washout period is 4 weeks. For patients receiving anticancer immunotherapy, such as CTGA-4 antagonists, the washout period is 6 weeks.

12. Active diarrhea CTCAE grade 2 or disease associated with chronic diarrhea (eg, irritable bowel syndrome, inflammatory bowel disease).

13. Presence 2: Grade 2 CTCAE toxicity (excluding alopecia, peripheral neuropathy, and ototoxicity, which are excluded if >= Grade 3 CTCAE) due to prior cancer therapy.

14. Active infection requiring systemic antibacterial therapy.

15. Active ulceration of the upper gastrointestinal tract or gastrointestinal bleeding.

16. Active bleeding diathesis or use of oral anti-vitamin K drugs (except low dose warfarin and aspirin or equivalent if INR <= 2.0).

17. Active autoimmune disease, motor neuropathy of autoimmune origin, and other autoimmune diseases of the central nervous system.

18. Patients requiring concomitant immunosuppressants or chronic corticosteroid treatment, except: steroid replacement in adrenal insufficiency

- 39 047973 topical, inhaled, nasal and ophthalmic steroids are permitted.

19. Use of any live vaccines against infectious diseases (eg, influenza, chickenpox, pneumococcus) within 4 weeks of starting study treatment (note: the use of live vaccines is not allowed throughout the study period).

20. Use of hematopoietic colony-stimulating growth factors (e.g., G-CSF, GM-CSF, M-CSF) < 2 weeks prior to initiation of study drug. An erythroid-stimulating agent is permitted if initiated at least 2 weeks prior to the first dose of study drug.

21. Major surgery within 2 weeks after administration of the first dose of study drug (NB mediastinoscopy, insertion of a central venous access device, or insertion of a feeding tube are not considered major surgery).

22. Radiation therapy within 2 weeks of the first dose of study drug, except for palliative, limited-field radiation therapy, such as for bone pain or a focal painful tumour. To be able to assess response to treatment, patients must have measurable disease that was not irradiated.

23. Participation in an intervention study within 2 weeks after the first dose of study drug.

24. Any medical condition that, in the opinion of the investigator, may interfere with the patient's participation in the clinical trial due to safety concerns, compliance with clinical trial procedures, or interpretation of trial results.

25. Sexually active men, unless they use a condom during intercourse while taking the drug and for 90 days after stopping study treatment, and must not have fathered a child during this period. Condoms should also be used by men who have had a vasectomy to prevent delivery of the drug via semen.

26. Pregnant or lactating women, for whom pregnancy is defined as the condition of the woman after conception and before termination of pregnancy, confirmed by a positive laboratory test for hCG. In rare cases of endocrine-secreting tumors, hCG levels may be higher than normal, but in the absence of pregnancy, the patient should have a repeat serum hCG test (without reporting the result) and a vaginal/pelvic ultrasound to exclude pregnancy. After confirmation of the results and discussion with the health care provider, these patients may participate in the study.

27. Women of childbearing potential, defined as all women physiologically capable of becoming pregnant, unless they used highly effective contraceptive methods during study treatment and for 90 days after the last dose of any study drug. Highly effective contraceptive methods include:

Total abstinence (when consistent with the patient's preferred and usual lifestyle. Periodic abstinence (e.g., calendar, ovulatory, symptothermal, postovulatory) and withdrawal are not acceptable methods of contraception.

Sterilization for women (presence of surgical bilateral oophorectomy with or without hysterectomy), total hysterectomy, or tubal ligation at least 6 weeks before receiving the study drug. In the case of oophorectomy alone, only if the woman's reproductive status is confirmed by subsequent hormone assessment.

Sterilization for men (at least 6 months prior to screening). For patients in the study, the male partner who had undergone a vasectomy must have been the patient's only partner.

Use of oral (estrogen and progestin), injectable, or implanted hormonal contraception or placement of an intrauterine device (IUD) or intrauterine system (IUS) or other forms of hormonal contraception that have comparable effectiveness (failure rate < 1%), such as a hormonal vaginal ring or transdermal hormonal contraception.

If using oral contraceptives, women should be stable on the same pill for at least 3 months before starting the study drug.

Women who are considered postmenopausal and not of childbearing potential if they have had natural (spontaneous) amenorrhea for 12 months with an appropriate clinical profile (e.g. age-appropriate, history of vasomotor symptoms) or if they have had bilateral surgical oophorectomy (with or without hysterectomy) or tubal ligation at least 6 weeks previously. In the case of oophorectomy alone, only if the woman's reproductive status is confirmed by subsequent hormone assessment is she considered not of childbearing potential.

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Dose-limiting toxicity and principles of dose modification.

Dose-limiting toxicity (DLT) is defined as any of the following events that are considered at least possibly related to the ADC in the investigator's judgment that occurs within the 21-day DLT assessment period. Toxicity clearly and directly related to the underlying disease or other etiology is excluded from this definition.

Definitions of DLT.

Hematological DLT is defined as:

Febrile neutropenia or neutropenic infection grade 3 or 4.

Grade 4 neutropenia lasting > 7 days.

Thrombocytopenia grade 4.

Grade 3 thrombocytopenia with clinically significant bleeding or grade 3 thrombocytopenia requiring platelet transfusion.

Anemia grade 3 requiring transfusion.

Anemia 4 degrees.

Non-hematological DLT is defined as:

Non-hematological toxicity grade 4.

Grade 3 non-hematological toxicity lasting >3 days despite optimal supportive care or medical intervention.

High's Law case (AST and/or ALT level > 3x ULN and bilirubin level > 2x ULN, and no initial evidence of cholestasis (serum alkaline phosphatase (ALP) activity < 2x ULN) and no other cause that could explain the combination of elevated serum transaminase and total bilirubin levels, such as viral hepatitis A, B, or C, pre-existing or acute liver disease, or another drug capable of causing the observed injury).

Grade 3 or higher hypersensitivity/infusion-related reaction (irrespective of premedication). Grade 3 hypersensitivity/infusion-related reaction that resolves within 8 hours of onset with appropriate clinical management does not qualify as DLT.

Decrease in LVEF to <40% or >20% of baseline.

Grade 4 tumor lysis syndrome (Grade 3 TLS will not be considered TLS unless it results in irreversible damage to the target organ).

The following conditions are not considered non-hematological DLT:

Fatigue grade 3 for < 7 days.

Grade 3 diarrhea, nausea, or vomiting in the absence of premedication that responds to therapy and improves by at least Grade 1 within 3 days for Grade 3 events or to < Grade 1 within 7 days.

An increase in AST or ALT > 5xULN but < 8xULN, without a concomitant increase in bilirubin that resolves to < grade 2 within 5 days of onset.

Serum lipase or serum amylase grade 3 for < 7 days in the absence of clinical signs or symptoms of pancreatitis.

Patients who experience DLT that resolves or stabilizes with appropriate medical management may continue treatment at the discretion of the investigator in consultation with the sponsor.

Dose modifications.

Recommendations for the management of specific toxicities are detailed in the table below. For the management of events not listed in the tables, the following recommendations may provide guidance to investigators:

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Rating NA ADC Management Guide 1 No dose adjustment is required. 2 First occurrence: Consider stopping one or both medications until improvement to < Grade 1 or to baseline. Up to 1 dose of one or both medications may be missed until improvement occurs. If improvement to < Grade 1 or to baseline occurs within 21 days after the last scheduled (but missed) dose of one or both medications, continue one or both medications at the originally scheduled dose level in subsequent treatment cycles. If improvement to < Grade 1 or to baseline does not occur within 21 days after the last scheduled (but missed) dose, permanently stop one or both medications. Second occurrence: Stop one or both medications until improvement to < Grade 1 or to baseline. Up to 1 dose of one or both medications may be missed until improvement occurs. If improvement to < Grade 1 or to baseline occurs within 21 days after the last scheduled (but missed) dose, continue one or both medications at 1 dose level below the originally scheduled dose in subsequent treatment cycles. If improvement to < Grade 1 or to baseline does not occur within 21 days after the last scheduled (but missed) dose, permanently stop one or both medications. Third occurrence: Permanently stop one or both medications. 3 First occurrence: Stop one or both medications until improvement to < Grade 1 or to baseline. Up to 1 dose of one or both medications may be skipped until improvement occurs, then continue at 1 dose below the original prescribed dose in subsequent treatment cycles. Second occurrence: Permanently stop one or both medications 4 Permanently stop taking one or both medications.

Example 3.

Methods.

MTT-based proliferation assay and IC50 calculation in cell lines exposed (96 h) to increasing concentrations of ADCx19. Pearson correlation (r): calculated for IC50 compared to cell surface CD19 expression levels (absolute fluorescence quantification with Quantum Simply Cellular beads; non-absolute, data from PMID 29298756) and compared to RNA levels (HT-12 Illumina arrays and HTG EdgeSeq Cancer Biomarker Panel, data from PMID 29066507). Synergy at 96 hours was assessed using the Chou-Talalay combination index (CI) (synergy CI <0.9, additive CI = 0.9-1.1, antagonism/no benefit CI >1.1) in 2 activated B-cell (ABC) DLBCL (OCI-LY-3, TMD8) and 2 germinal center (GCB) DLBCL (VAL, WSU-DLCL2).

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Results.

The median IC50 of ADCx19 was 4 pM (95% CI, 2-10 pM) in 48 B-cell lymphoma lines and, as expected based on the CD19 expression profile, was more than 800-fold higher in 9 T-cell lymphoma lines (3.5 nM; 95% CI, 0.8-11 nM). Focusing on B-cell lymphomas, ADCx19 activity in vitro correlated with its target expression measured at both the cell surface protein [(absolute quantification, n = 40, r -0.37 P < 0.02; non-absolute quantification, n = 42, -0.48, P < 0.001] and RNA [(arrays, n = 39, -0.69 P < 0.001; HTG, n = 31, -0.73 P < 0.001] levels. In DLBCL, the presence of BCL2 and MYC translocations or TP53 inactivation did not affect ADCx19 sensitivity.

ADCx19 was then combined in GCB- and ABC-DLBCL cell lines with one or the other agent: PI3K inhibitor, idelalisib, and copanlisib. Synergy in all cell lines was achieved when ADCx19 was combined with idelalisib. Synergy was observed in half of the cell lines tested with copanlisib (OCI-LY-3, VAL).

The data is presented in the tables below.

Combinations of ADCx19 + idelalisib.

Cell line: OCI-LY3.

Cell Access Identifier RRID: CVCL_8800.

ADCxl9 (pm) Idelalisib (nM) Fa Di ADCxl9 (pM) Idelalisib (nM) Fa DI 0,02 4.88 0.95 0.95 1.56 1250 0.46 0.16 0,02 19.53 0.85 0.21 1.56 5000 0.36 0.22 0,02 78,13 0.78 0.26 1.56 20000 0.28 0.38 0,02 312,5 0.80 1.36 6.25 4.88 0.64 0,04 0,02 1250 0.68 1.29 6.25 19.53 0.62 0,04 0,02 5000 0.55 1.46 6.25 78,13 0.53 0,04 0,02 20000 0.39 1,21 6.25 312,5 0.44 0,05 0,1 4.88 1,00 269,02 6.25 1250 0.37 0,08 0,1 19.53 0.95 2.79 6.25 5000 0.26 0.09 0,1 78,13 0.73 0,15 6.25 20000 0.18 0,10 0,1 312,5 0.72 0,50 25 4.88 0.41 0,08 0,1 1250 0.65 0.97 25 19.53 0.33 0,06 0,1 5000 0.52 1.04 25 78,13 0.25 0,05 0,1 20000 0.36 0.89 25 312,5 0,20 0,04 0.39 4.88 0.88 0,10 25 1250 0,12 0,03 0.39 19.53 0.93 1.53 25 5000 0.11 0,03 0.39 78,13 0.75 0.18 25 20000 0,08 0,03 0.39 312,5 0.64 0,20 100 4.88 0,15 0.13 0.39 1250 0,50 0.22 100 19.53 0.09 0.09 0.39 5000 0.46 0.59 100 78,13 0,06 0,07 0.39 20000 0.33 0.71 100 312,5 0,05 0,06 1.56 4.88 0.83 0,05 100 1250 0,05 0,06 1.56 19.53 0.74 0,05 100 5000 0,04 0,05 1.56 78,13 0.64 0,06 100 20000 0,03 0,04 1.56 312,5 0.55 0.09

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Cell line: TMD8.

Cell Access Identifier RRID: CVCL_A442.

Reference: Tohda et al., Leuk. Res. 30:1385-1390(2006).

ADCxl9 (pm) Idelalisib (nM) Fa DI ADCxl9 (pM) Idelalisib (nM) Fa DI 0,02 4.88 0.85 1.47 0.39 78,13 0.27 0.83 0,02 19.53 0.62 1.27 0.39 312,5 0,10 0.77 0,02 78,13 0.31 1.06 1.56 4.88 0.64 0.84 0,02 312,5 0.13 1.08 1.56 19.53 0.44 0.64 ο,ι 4.88 0.85 1.61 1.56 78,13 0.16 0.37 ο,ι 19.53 0.62 1.29 1.56 312,5 0,06 0.37 ο,ι 78,13 0.29 0.97 6.25 4.88 0.25 0.17 ο,ι 312,5 0,12 1,02 6.25 19.53 0,12 0.09 0.39 4.88 0.81 1.58 6.25 78,13 0,04 0,05 0.39 19.53 0.61 1.29 6.25 312,5 0,02 0,07

Cell line: VAL.

Cell Access Identifier RRID: CVCL_1819.

ADCxl9 (pm) Idelalisib (nM) Fa DI ADCxl9 (pM) Idelalisib (nM) Fa DI 0,02 4.88 0.92 0.33 0.39 4.88 0.57 0.69 0,02 19.53 0.88 0.47 0.39 19.53 0.52 0.66 0,02 78,13 0.78 0.32 0.39 78,13 0,50 0.66 0,02 312,5 0.78 1D4 0.39 312,5 0,50 0.69 0,02 1250 0.72 1.94 0.39 1250 0.48 0.78 0,02 5000 0.55 1.25 0.39 5000 0,40 0.87 0,02 20000 0.38 0.90 0.39 20000 0,30 0.90 0,1 4.88 0.76 0.23 1.56 4.88 0.28 2.07 0,1 19.53 0,70 0.23 1.56 19.53 0.27 2.04 0,1 78,13 0.67 0.26 1.56 78,13 0.29 2,10 0,1 312,5 0.69 0.54 1.56 312,5 0.28 2.09 0,1 1250 0.63 0.86 1.56 1250 0.28 2.09 0,1 5000 0.51 1.03 1.56 5000 0.27 2.11 0,1 20000 0.36 0.91 1.56 20000 0.23 2.09

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Cell line: WSU-DLCL2.

Cell Access Identifier RRID: CVCL_1902.

ADCxl9 (pm) Idelalisib (nM) Fa DI ADCxl9 (pM) Idelalisib (nM) Fa DI 0,02 4.88 0.91 0.17 1.56 1250 0.49 0.27 0,02 19.53 0.87 0.23 1.56 5000 0.37 0.25 0,02 78,13 0.84 0.52 1.56 20000 0.24 0,20 0,02 312,5 0.81 1.33 6.25 4.88 0,50 0.59 0,02 1250 0.73 1.57 6.25 19.53 0.46 0.54 0,02 5000 0.57 1,15 6.25 78,13 0.42 0,50 0,02 20000 0.31 0.29 6.25 312,5 0,40 0.48 0,1 4.88 0.89 0.14 6.25 1250 0.34 0.43 0,1 19.53 0.78 0,07 6.25 5000 0.28 0.39 0,1 78,13 0.79 0.24 6.25 20000 0.17 0.29 0,1 312,5 0.76 0.62 25 4.88 0.31 1.52 0,1 1250 0.66 0.69 25 19.53 0.33 1.59 0,1 5000 0.53 0.75 25 78,13 0.31 1.50 0,1 20000 0.32 0.36 25 312,5 0.29 1.43 0.39 4.88 0.84 0.13 25 1250 0.26 1.32 0.39 19.53 0.79 0.13 25 5000 0.21 1,14 0.39 78,13 0.75 0.19 25 20000 0.14 0.88 0.39 312,5 0.72 0.42 100 4.88 0.26 5.29 0.39 1250 0.62 0.49 100 19.53 0.25 5.16 0.39 5000 0,50 0.56 100 78,13 0.25 5.18 0.39 20000 0.28 0.24 100 312,5 0.26 5.20 1.56 4.88 0.66 0.22 100 1250 0.23 4.80 1.56 19.53 0.63 0.21 100 5000 0.19 4.25 1.56 78,13 0.59 0,20 100 20000 0,12 3.10 1.56 312,5 0.56 0.23

- 45 047973

Combinations of ADCx19 + copanlisib.

Cell line: OCI-LY3.

Cell Access Identifier RRID: CVCL_8800.

ADCxl9 (pm) Copanlisib (nM) Fa DI ADCxl9 (pM) Copanlisib (nM) Fa DI 0,02 0.49 1 3479949534 1.56 31.25 0.43 0.41 0,02 1.95 1 3482371896 1.56 125 0.25 0.63 0,02 7.81 0.85 0.68 1.56 500 0.09 0.77 0,02 31.25 0.59 0.67 6.25 0.49 0.63 0.95 0,02 125 0.34 0.98 6.25 1.95 0,50 0.43 0,02 500 0,10 0.85 6.25 7.81 0.43 0.35 0,1 0.49 0.95 0.68 6.25 31.25 0.25 0.23 0,1 1.95 0.91 0.53 6.25 125 0,15 0.35 ο,ι 7.81 0.78 0.46 6.25 500 0,07 0.54 ο,ι 31.25 0.51 0.49 25 0.49 0.45 1.23 ο,ι 125 0.33 0.92 25 1.95 0.35 0.65 ο,ι 500 0,10 0.87 25 7.81 0.22 0.26 0.39 0.49 0.99 20.88 25 31.25 0.11 0,12 0.39 1.95 0.92 1.42 25 125 0,07 0.18 0.39 7.81 0.67 0.32 25 500 0,04 0.34 0.39 31.25 0.46 0.42 100 0.49 0.18 0,60 0.39 125 0.29 0.78 100 1.95 0.11 0.26 0.39 500 0,10 0.78 100 7.81 0,06 0.09 1.56 0.49 0.75 0.58 100 31.25 0,03 0,05 1.56 1.95 0.72 0.51 100 125 0,03 0.09 1.56 7.81 0.59 0.34 100 500 0,03 0.21

Cell line: TMD8.

Cell Access Identifier RRID: CVCL_A442.

Reference: Tohda et al., Leuk. Res. 30:1385-1390(2006).

ADCxl9 (pM) Copanlisib (nM) Fa Di ADCxl9 (pM) Copanlisib (nM) Fa DI 0,02 0.49 0.83 1.30 0.39 1.95 0.37 1.05 0,02 1.95 0.42 1.09 1.56 0.49 0.43 0.91 0,1 0.49 0.80 1.23 1.56 1.95 0.14 0.51 0,1 1.95 0.41 1.08 6.25 0.49 0.09 0.55 0.39 0.49 0.73 1,20 6.25 1.95 0,03 0.24

Cell line: VAL.

Cell Access Identifier RRID: CVCL 1819.

ADCxl9 (pM) Copanlisib (nM) Fa DI ADCxl9 (pM) Copanlisib (nM) Fa DI 0,02 0.49 0.80 0.58 oh,1 31.25 0.39 0.84 0,02 1.95 0.73 0.64 0,1 125 0.26 1,11 0,02 7.81 0.57 0.61 0.39 0.49 0.42 0.94 0,02 31.25 0.44 0.93 0.39 1.95 0.38 0.83 0,02 125 0.28 1,18 0.39 7.81 0.34 0.75 0,1 0.49 0.67 0.90 0.39 31.25 0,30 0.86 0,1 1.95 0.57 0.62 0.39 125 0.23 1.06 0,1 7.81 0.48 0.63

- 46 047973

Cell line: WSU-DLCL2.

Cell Access Identifier RRID: CVCL_1902.

ADCxl9 (pM) Copanlisib (nM) Fa DI ADCxl9 (pm) Copanlisib (nM) Fa DI 0,02 0.49 1 1.25367E+11 1.56 31.25 0.48 0.72 0,02 1.95 1 1.25367E+11 1.56 125 0.26 1.71 0,02 7.81 0.91 0.38 6.25 0.49 0.52 1,02 0,02 31.25 0.74 0.73 6.25 1.95 0.48 0.82 0,02 125 0.35 1.86 6.25 7.81 0.43 0,70 0,1 0.49 1 6.26833E+11 6.25 31.25 0.37 0.84 0,1 1.95 0.98 15.34 6.25 125 0,20 1.62 ο,ι 7.81 0.83 0.42 25 0.49 0.39 1.70 ο,ι 31.25 0.70 0.74 25 1.95 0.38 1.56 ο,ι 125 0.32 1.80 25 7.81 0.36 1.44 0.39 0.49 0.93 4.25 25 31.25 0.31 1.39 0.39 1.95 0.87 1.51 25 125 0.17 1.72 0.39 7.81 0.75 0.55 100 0.49 0.32 3.86 0.39 31.25 0.62 0.74 100 1.95 0.31 3.73 0.39 125 0.32 1.81 100 7.81 0.29 3.13 1.56 0.49 0.75 1.53 100 31.25 0.28 3.21 1.56 1.95 0.66 0.74 100 125 0,15 2,16 1.56 7.81 0.59 0.57

Conclusion.

The potent in vitro anti-lymphoma activity of single agent ADCx19 correlates with its target expression and supports ongoing clinical trials in relapsed/refractory DLBCL. Data on the novel combination of idelalisib and copanlisib suggest plausible clinical synergy.

Example 4.

Methods.

MTT-based proliferation assay and IC50 calculation in cell lines exposed (96 h) to increasing concentrations of ADCx19. Pearson correlation (r): calculated for IC50 compared to cell surface CD19 expression levels (absolute fluorescence quantification with Quantum Simply Cellular beads; non-absolute, data from PMID 29298756) and compared to RNA levels (HT-12 Illumina arrays and HTG EdgeSeq Cancer Biomarker Panel, data from PMID 29066507). Synergy at 96 hours was assessed using the Chou-Talalay combination index (CI) (synergy CI <0.9, additive CI = 0.9-1.1, antagonism/no benefit CI >1.1) in 2 activated B-cell (ABC) DLBCL (OCI-LY-3, TMD8) and 2 germinal center (GCB) DLBCL (VAL, WSU-DLCL2).

Results.

The median IC50 of ADCx19 was 4 pM (95% CI, 2-10 pM) in 48 B-cell lymphoma lines and, as expected based on the CD19 expression profile, was more than 800-fold higher in 9 T-cell lymphoma lines (3.5 nM; 95% CI, 0.8-11 nM). Focusing on B-cell lymphomas, ADCx19 activity in vitro correlated with its target expression measured at both the cell surface protein [(absolute quantification, n = 40, r -0.37 P < 0.02; non-absolute quantification, n = 42, -0.48, P < 0.001] and RNA [(arrays, n = 39, -0.69 P < 0.001; HTG, n = 31, -0.73 P < 0.001] levels. In DLBCL, the presence of BCL2 and MYC translocations or TP53 inactivation did not affect ADCx19 sensitivity.

ADCx19 was then combined in GCB- and ABC-DLBCL cell lines with the proteasome inhibitor bortezomib (ABC only), the chemotherapeutic agent bendamustine, and the PARP inhibitor olaparib.

Synergism was achieved in all cell lines except OCI-L Y-3 with bendamustine.

Synergy was observed in half of the cell lines tested with olaparib (VAL, WSUDLCL2).

No benefit was observed with the addition of bortezomib and lenalidomide to ADCT-402 in two ABC DLBCL (TMD8, OCI-LY-3).

The data is presented in the tables below.

- 47 047973

ADCx19 + bendamustine combinations.

Cell line: OCI-LY3.

Cell Access Identifier RRID: CVCL_8800.

ADCxl9 (pm) Bendamustine (nM) Fa DI ADCxl9 (pM) Bendamustine (nM) Fa di 0,02 4.88 0.99 4.06 1.56 312,5 0.82 1.52 0,02 19.53 0.90 0,07 1.56 1250 0.87 2.91 0,02 78,13 1,00 2.53E+21 1.56 5000 0.71 0.61 0,02 312,5 1,00 1.01E+22 6.25 4.88 0.55 0.78 0,02 1250 0.99 959,02 6.25 19.53 0.55 0.80 0,02 5000 0.88 1.79 6.25 78,13 0.59 1,02 0,1 4.88 0.99 17.12 6.25 312,5 0,60 1,10 0,1 19.53 0.99 46.93 6.25 1250 0.61 1D2 0,1 78,13 0.97 5.03 6.25 5000 0.42 0.35 0,1 312,5 0.95 2.89 25 4.88 0.32 0.73 0,1 1250 0.97 45.19 25 19.53 0.32 0.74 0,1 5000 0.83 0,60 25 78,13 0.31 0.69 0.39 4.88 0.96 4.86 25 312,5 0.31 0,70 0.39 19.53 0.87 0.68 25 1250 0.28 0.57 0.39 78,13 0.89 0.99 25 5000 0.16 0.18 0.39 312,5 1,00 1.01E+22 100 4.88 0.11 0.36 0.39 1250 0.99 1028,40 100 19.53 0.11 0.39 0.39 5000 0.83 0.94 100 78,13 0.11 0.38 1.56 4.88 0.81 1.29 100 312,5 0,10 0.33 1.56 19.53 0.86 2.31 100 1250 0,10 0,30 1.56 78,13 0.82 1.47 100 5000 0,06 0.14

- 48 047973

Cell line: TMD8.

Cell Access Identifier RRID: CVCL_A442.

Reference: Tohda et al., Leuk. Res. 30:1385-1390(2006).

ADCxl9 (pm) Bendamustine (nM) Fa DI ADCxl9 (pM) Bendamustine (nM) Fa DI 0,02 4.88 0.92 0,07 1.56 1250 0.94 12.60 0,02 19.53 0.92 0.09 1.56 5000 0.88 4.76 0,02 78,13 0.93 0,30 1.56 20000 0.27 0,04 0,02 312,5 0.97 10,16 6.25 4.88 0.69 1.92 0,02 1250 0.94 6.75 6.25 19.53 0.71 2.24 0,02 5000 0.87 1.46 6.25 78,13 0,70 2.04 0,02 20000 0.31 0,00 6.25 312,5 0.66 1.59 0,1 4.88 0.90 0.24 6.25 1250 0.68 1.90 0,1 19.53 0.90 0.24 6.25 5000 0.56 0.88 ο,ι 78,13 0.92 0.44 6.25 20000 0,12 0,04 ο,ι 312,5 0.91 0.53 25 4.88 0.27 0.61 ο,ι 1250 0.96 24.62 25 19.53 0.26 0.58 ο,ι 5000 0.86 1,22 25 78,13 0.26 0,60 ο,ι 20000 0.32 0,01 25 312,5 0.26 0.57 0.39 4.88 0.95 2.67 25 1250 0.25 0.54 0.39 19.53 0.94 2.26 25 5000 0,20 0.35 0.39 78,13 0.94 2.08 25 20000 0,04 0,03 0.39 312,5 0.97 17.42 100 4.88 0,08 0.33 0.39 1250 0.99 702.11 100 19.53 0,08 0.34 0.39 5000 0.93 12.28 100 78,13 0,08 0.35 0.39 20000 0,30 0,01 100 312,5 0,08 0.31 1.56 4.88 0.92 4.65 100 1250 0,08 0.31 1.56 19.53 0.94 8.37 100 5000 0,06 0.19 1.56 78,13 0.91 4.10 100 20000 0,01 0,03 1.56 312,5 0.92 6.09

Cell line: VAL.

Cell Access Identifier RRID: CVCL_1819.

ADCxl9 (pm) Bendamustine (nM) Fa DI ADCxl9 (pM) Bendamustine (nM) Fa DI 0,02 4.88 0.89 0.58 0.39 4.88 0.64 1.47 0,02 19.53 0.87 0.55 0.39 19.53 0,60 1,15 0,02 78,13 0.88 1,10 0.39 78,13 0.63 1.40 0,02 312,5 0.84 1.44 0.39 312,5 0.58 1.08 0,02 1250 0.82 3.09 0.39 1250 0.58 1,11 0,02 5000 0.69 1.49 0.39 5000 0.46 0.65 0,02 20000 0.26 0,03 0.39 20000 0.16 0,08 0,1 4.88 0.82 1.27 1.56 4.88 0.24 0.62 0,1 19.53 0.79 1,01 1.56 19.53 0.24 0.62 0,1 78,13 0.80 1,16 1.56 78,13 0.27 0.73 0,1 312,5 0.77 1.09 1.56 312,5 0.22 0.52 0,1 1250 0.75 1.60 1.56 1250 0.22 0.51 0,1 5000 0.61 0.77 1.56 5000 0.17 0.34 0,1 20000 0.24 0,06 1.56 20000 0,07 0.09

- 49 047973

Cell line: WSU-DLCL2.

Cell Access Identifier RRID: CVCL_1902.

ADCxl9 (pm) Bendamustine (nM) Fa DI ADCxl9 (pM) Bendamustine (nM) Fa DI 0,02 4.88 0.92 0.13 1.56 1250 0.86 2.68 0,02 19.53 0.91 0,07 1.56 5000 0.90 5.05 0,02 78,13 0.91 0.13 1.56 20000 0.53 0,12 0,02 312,5 0.90 0,07 6.25 4.88 0,70 1.79 0,02 1250 0.92 1.69 6.25 19.53 0.67 1.37 0,02 5000 0.92 49.68 6.25 78,13 0.66 1.31 0,02 20000 0.88 0,06 6.25 312,5 0.67 1.40 0,1 4.88 0.93 1.50 6.25 1250 0,70 1.74 0,1 19.53 0.90 0.36 6.25 5000 0.58 0.71 0,1 78,13 0.89 0.26 6.25 20000 0.31 0.09 0,1 312,5 0.90 0,30 25 4.88 0.38 0.62 0,1 1250 0.89 0.25 25 19.53 0.35 0.51 0,1 5000 0.92 63.54 25 78,13 0.37 0,60 0,1 20000 0.84 0.11 25 312,5 0.37 0.57 0.39 4.88 0.88 0.84 25 1250 0.35 0.52 0.39 19.53 0.87 0.77 25 5000 0.29 0,30 0.39 78,13 0.87 0.76 25 20000 0.25 0.22 0.39 312,5 0.89 0.97 100 4.88 0.22 0.61 0.39 1250 0.87 0.73 100 19.53 0.23 0.69 0.39 5000 0.90 1.27 100 78,13 0.23 0.66 0.39 20000 0.75 0.18 100 312,5 0.22 0.62 1.56 4.88 0.90 4.65 100 1250 0.21 0.58 1.56 19.53 0.87 2.76 100 5000 0.19 0.47 1.56 78,13 0.86 2.44 100 20000 0.13 0,20 1.56 312,5 0.85 2.34

- 50 047973

Combinations of ADCx19 + olaparib.

Cell line: OCI-LY3.

Cell Access Identifier RRID: CVCL_8800.

ADCxl9 (nM) Olaparib (nM) Fa DI ADCxl9 (pm) Olaparib (nM) Fa DI 0,02 27,43 0.96 0,08 1.56 740,74 0.79 1.34 0,02 82.30 1 338.21 1.56 2222,22 0.44 2.07 0,02 246.91 1 365,58 6.25 27,43 0.78 1.31 0,02 740,74 0.92 1.41 6.25 82.30 0.69 1,01 0,02 2222,22 0,60 2.33 6.25 246.91 0.54 0.85 0,1 27,43 0.96 0.13 6.25 740,74 0.37 0.98 0,1 82.30 0.94 0.22 6.25 2222,22 0.33 2.06 0,1 246.91 0.96 0.64 25 27,43 0.42 1.79 0,1 740,74 0.95 1.64 25 82.30 0.41 1.78 0,1 2222,22 0.63 2.43 25 246.91 0.28 1.36 0.39 27,43 0.91 0.21 25 740,74 0.34 2,00 0.39 82.30 0.95 0.42 25 2222,22 0.16 2.06 0.39 246.91 0.94 0.71 100 27,43 0.18 3.23 0.39 740,74 0.84 1,21 100 82.30 0.17 3.04 0.39 2222,22 0.54 2.22 100 246.91 0.14 2.82 1.56 27,43 0.77 0.33 100 740,74 0.13 2.82 1.56 82.30 0.83 0.51 100 2222,22 0,05 2,18 1.56 246.91 0.84 0.77

Cell line: TMD8.

Cell Access Identifier RRID: CVCL_A442.

Reference: Tohda et al., Leuk. Res. 30:1385-1390(2006).

ADCxl9 (pM) Olaparib (nM) Fa DI ADCxl9 (pM) Olaparib (nM) Fa DI 0,02 4.88 0.85 1.48 0.39 78,13 0.27 0.82 0,02 19.53 0.62 1.26 1.56 4.88 0.64 0.84 0,02 78,13 0.31 1.05 1.56 19.53 0.44 0.64 0,1 4.88 0.85 1.62 1.56 78,13 0.16 0.37 0,1 19.53 0.62 1.29 6.25 4.88 0.25 0.17 0,1 78,13 0.29 0.95 6.25 19.53 0,12 0.09 0.39 4.88 0.81 1.58 6.25 78,13 0,04 0,05 0.39 19.53 0.61 1.28

- 51 047973

Cell line: VAL.

Cell Access Identifier RRID: CVCL_1819.

ADCxl9 (pm) Olaparib (nM) Fa Di ADCxl9 (pM) Olaparib (nM) Fa DI 0,02 4.88 0.92 0.33 0.39 4.88 0.57 0.69 0,02 19.53 0.88 0.47 0.39 19.53 0.52 0.66 0,02 78,13 0.78 0.32 0.39 78,13 0,50 0.66 0,02 312,5 0.78 1,14 0.39 312,5 0,50 0.69 0,02 1250 0.72 1.94 0.39 1250 0.48 0.78 0,02 5000 0.55 1.25 0.39 5000 0,40 0.87 0,02 20000 0.38 0.90 0.39 20000 0,30 0.90 0,1 4.88 0.76 0.23 1.56 4.88 0.28 2.07 0,1 19.53 0,70 0.23 1.56 19.53 0.27 2.04 0,1 78,13 0.67 0.26 1.56 78,13 0.29 2,10 0,1 312,5 0.69 0.54 1.56 312,5 0.28 2.09 0,1 1250 0.63 0.86 1.56 1250 0.28 2.09 0,1 5000 0.51 1.03 1.56 5000 0.27 2.11 0,1 20000 0.36 0.91 1.56 20000 0.23 2.09

Cell line: WSU-DLCL2.

Cell Access Identifier RRID: CVCL_1902.

ADCxl9 (pM) Olaparib (nM) Fa DI ADCxl9 (pM) Olaparib (nM) Fa DI 0,02 4.88 0.91 0.17 1.56 1250 0.49 0.27 0,02 19.53 0.87 0.23 1.56 5000 0.37 0.25 0,02 78,13 0.84 0.52 1.56 20000 0.24 0,20 0,02 312,5 0.81 1.33 6.25 4.88 0,50 0.59 0,02 1250 0.73 1.57 6.25 19.53 0.46 0.54 0,02 5000 0.57 1,15 6.25 78,13 0.42 0,50 0,02 20000 0.31 0.29 6.25 312,5 0,40 0.48 0,1 4.88 0.89 0.14 6.25 1250 0.34 0.43 0,1 19.53 0.78 0,07 6.25 5000 0.28 0.39 0,1 78,13 0.79 0.24 6.25 20000 0.17 0.29 0,1 312,5 0.76 0.62 25 4.88 0.31 1.52 0,1 1250 0.66 0.69 25 19.53 0.33 1.59 0,1 5000 0.53 0.75 25 78,13 0.31 1.50 0,1 20000 0.32 0.36 25 312,5 0.29 1.43 0.39 4.88 0.84 0,13 25 1250 0.26 1.32 0.39 19.53 0.79 0,13 25 5000 0.21 1,14 0.39 78,13 0.75 0.19 25 20000 0.14 0.88 0.39 312,5 0.72 0.42 100 4.88 0.26 5.29 0.39 1250 0.62 0.49 100 19.53 0.25 5.16 0.39 5000 0,50 0.56 100 78,13 0.25 5.18 0.39 20000 0.28 0.24 100 312,5 0.26 5.20 1.56 4.88 0.66 0.22 100 1250 0.23 4.80 1.56 19.53 0.63 0.21 100 5000 0.19 4.25 1.56 78,13 0.59 0,20 100 20000 0,12 3.10 1.56 312,5 0.56 0.23 1.56 1250 0.49 0.27

- 52 047973

ADCx19 + bortezomib combinations.

Cell line: OCI-LY3.

Cell Access Identifier RRID: CVCL_8800.

ADCxl9 (nM) Bortezomib (nM) Fa DI ADCxl9 (pm) Bortezomib (nM) Fa DI 0,02 0,002743 1 11,0 1.56 0,074074 1 857,1 0,02 0,008230 1 11,0 1.56 0,222222 0.927 127.2 0,02 0,024691 1 11,1 1.56 0,666667 0.974 275.6 0,02 0,074074 1 11.3 6.25 0,002743 0.853 2,2 0,02 0,222222 1 12,0 6.25 0,008230 0,865 6.0 0,02 0,666667 1 14.1 6.25 0,024691 0.979 10.1 0,1 0,002743 1 54.9 6.25 0,074074 0,883 49.9 0,1 0,008230 1 55,0 6.25 0,222222 0,786 185.5 0,1 0,024691 1 55,0 6.25 0,666667 0,841 498.6 0,1 0,074074 1 55.3 25 0,002743 0,665 3.1 0,1 0,222222 1 56,0 25 0,008230 0,693 8.3 0,1 0,666667 1 58.1 25 0,024691 0,809 20.3 0.39 0,002743 1 214.2 25 0,074074 0,800 60.8 0.39 0,008230 1 214.2 25 0,222222 0,711 209.7 0.39 0,024691 1 214.3 25 0,666667 0,741 601,0 0.39 0,074074 1 214.5 100 0,002743 0,473 4,4 0.39 0,222222 1 215.2 100 0,008230 0,580 10.3 0.39 0,666667 1 217.3 100 0,024691 0.559 29,5 1.56 0,002743 0,980 1,2 100 0,074074 0,633 78.9 1.56 0,008230 0.998 2,1 100 0,222222 0,574 252.2 1.56 0,024691 1 856.9 100 0,666667 0.559 768,7

Cell line: TMD8.

Cell Access Identifier RRID: CVCL_A442.

Reference: Tohda et al., Leuk. Res. 30:1385-1390(2006).

ADCxl9 (pm) Bortezomib (nM) Fa DI ADCxl9 (pM) Bortezomib (nM) Fa DI 0,02 0,002743 1,00 0.81 1.56 0,002743 0.98 0.42 0,02 0,008230 1,00 1.74 1.56 0,008230 1,00 28,02 0,02 0,024691 1,00 4.54 1.56 0,024691 0.92 0.33 0,02 0,074074 1,00 12.93 1.56 0,074074 0.92 0.44 0,02 0,222222 1,00 38,10 1.56 0,222222 0.89 0.72 0,02 0,666667 0.79 1,16 1.56 0,666667 0.65 1D2 0,1 0,002743 1,00 2.17 6.25 0,002743 0.52 0.55 0,1 0,008230 1,00 3.10 6.25 0,008230 0.48 0.53 0,1 0,024691 1,00 5.90 6.25 0,024691 0,50 0.56 0,1 0,074074 1,00 14.29 6.25 0,074074 0.46 0.59 0,1 0,222222 0.99 0.86 6.25 0,222222 0.45 0.76 0,1 0,666667 0.73 1.08 6.25 0,666667 0.35 1,14 0.39 0,002743 1,00 7,12 25 0,002743 0,10 1,13 0.39 0,008230 1,00 8.05 25 0,008230 0.09 1,10 0.39 0,024691 1,00 10.85 25 0,024691 0,10 1,13 0.39 0,074074 0.97 0.33 25 0,074074 0,10 1,17 0.39 0,222222 0.98 0.85 25 0,222222 0.11 1.32 0.39 0,666667 0.72 1,10 25 0,666667 0,07 1.44

- 53 047973

Combinations of ADCx19 + lenalidomide.

Cell line: OCI-LY3.

Cell Access Identifier RRID: CVCL_8800.

ADCxl9 (pm) Lenalidomide (nM) Fa DI ADCxl9 (pM) Lenalidomide (nM) Fa DI 0,02 312 1 1.50E+22 1.56 5000 0.95 35.91 0,02 625 1 1.50E+22 1.56 10000 0.98 1218,24 0,02 1250 0.99 54.36 1.56 20000 0.82 0.97 0,02 2500 0.96 1.75 6.25 312 0.91 22.38 0,02 5000 0.92 0.33 6.25 625 0.84 2.75 0,02 10000 1 1.50E+22 6.25 1250 0.79 0.81 0,02 20000 0.84 0,60 6.25 2500 0.82 1.60 0,1 312 1 7.49E+22 6.25 5000 0.80 1.06 0,1 625 0.93 1.04 6.25 10000 0.85 3.46 0,1 1250 0.90 0.27 6.25 20000 0.71 0.64 0,1 2500 0.86 0,15 25 312 0.84 10.78 0,1 5000 0.81 0.16 25 625 0.77 2.21 0,1 10000 0.98 75.38 25 1250 0.81 4.91 0,1 20000 0.90 0.95 25 2500 0.73 1.25 0.39 312 1 2.92E+23 25 5000 0.77 2.27 0.39 625 0.88 0.56 25 10000 0,70 0.94 0.39 1250 0.97 97.25 25 20000 0.73 1.58 0.39 2500 0.81 0,15 100 312 0.52 0.22 0.39 5000 0.95 8.59 100 625 0.51 0.19 0.39 10000 0.97 83,90 100 1250 0.52 0.24 0.39 20000 0.84 0.75 100 2500 0,50 0,20 1.56 312 1 1D7E+24 100 5000 0.43 0.13 1.56 625 0.95 43.61 100 10000 0.48 0.29 1.56 1250 0.90 4.18 100 20000 0.45 0.39 1.56 2500 0.78 0.25

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Claims (2)

Cell line: TMD8. Cell RRID accession ID: CVCL_A442. Reference: Tohda et al., Leuk. Res. 30:1385–1390(2006). ADCxl9 (pM) Lenalidomide (nM) Fa CI ADCxl9 (pM) Lenalidomide (nM) Fa CI 0.02 312 0.85 3.13 1.56 312 0.80 1.84 0.02 625 0.75 2.16 1.56 625 0.69 1.38 0.02 1250 0.68 2.44 1.56 1250 0.63 1.73 0.02 2500 0.61 2.92 1.56 2500 0.54 1.91 0.02 5000 0.53 3.46 1.56 5000 0.45 2.08 0.02 10000 0.41 3.01 1.56 10000 0.37 2.30 0.02 20000 0.22 1.39 1.56 20000 0.18 0.90 0.1 312 0.83 2.66 6.25 312 0.47 0.28 0.1 625 0.74 2.05 6.25 625 0.42 0.32 0.1 1250 0.68 2.49 6.25 1250 0.39 0.45 0.1 2500 0.59 2.69 6.25 2500 0.32 0.50 0.1 5000 0.51 3.05 6.25 5000 0.29 0.71 0.1 10000 0.41 3.02 6.25 10000 0.20 0.66 0.1 20000 0.21 1.23 6.25 20000 0.11 0.39 0.39 312 0.82 2.36 25,312 0.21 0.38 0.39 625 0.78 2.92 25,625 0.19 0.37 0.39 1250 0.67 2.36 25 1250 0.16 0.35 0.39 2500 0.59 2.55 25 2500 0.13 0.35 0.39 5000 0.49 2.72 25 5000 0.10 0.34 0.39 10000 0.40 2.78 25 10000 0.10 0.39 0.39 20000 0.20 1.09 25 20000 0.04 0.26 Conclusion: The potent in vitro anti-lymphoma activity of single agent ADCx19 correlates with its target expression and supports ongoing clinical trials in relapsed/refractory DLBCL. Data with the novel combination of idelalisib and copanlisib indicate plausible clinical synergy. CLAIM 1. A method of treating cancer in an individual, comprising administering to the individual an effective amount of an anti-CD19 ADC and a PI3K inhibitor; and the said anti-CD19 ADC has the chemical structure: O where Ab is an antibody that binds to CD19. 2. The method according to claim 1, characterized in that the anti-CD19 ADC comprises an antibody having: (i) a VH domain having a sequence according to any of SEQ ID NO. 1, 2, 3, 4, 5 or 6, and a VL domain having a sequence according to any of SEQ ID NO. 7, 8, 9, 10, 11 or 12; (ii) a VH domain having the sequence according to SEQ ID NO. 1 and a VL domain having the sequence according to SEQ ID NO. 7; -