WO2024221187A1

WO2024221187A1 - Heteromultimer polypeptides

Info

Publication number: WO2024221187A1
Application number: PCT/CN2023/090376
Authority: WO
Inventors: Tong Zhang; Wei Jiang; Xiaoyun Guo
Original assignee: Biomap Intelligence Technology Sg Pte.Ltd.
Priority date: 2023-04-24
Filing date: 2023-04-24
Publication date: 2024-10-31

Abstract

Provided are heteromultimer polypeptides comprising a heterodimeric polypeptide complex and methods of production favoring heterodimeric polypeptide complex formation. Heterodimeric polypeptide complexes comprise two polypeptide immunoglobulin CH3 domains associated together, where each the region contains one or more mutations favoring heterodimer formation. Heteromultimer complexes can be used, for example, to facilitate the production of multispecific binding proteins.

Description

HETEROMULTIMER POLYPEPTIDES

FIELD OF INVENTION

The present invention relates to heteromultimer polypeptides comprising a heterodimeric polypeptide complex and methods of production favoring heterodimeric polypeptide complex formation.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The contents of the electronic sequence listing (MTP230151. xml; Size: 24, 308 bytes; and Date of Creation: April 23, 2023) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Conventional antibodies generally comprise two antigen binding domains able to bind to a particular epitope. The binding domains are provided heavy chain variable regions or a combination of heavy and light chain variable regions. Naturally occurring human antibodies are a multimer generally comprising two identical heavy chains and two identical light chains. Each heavy chain and light chain provide the same variable regions, resulting in a monospecific antibody. Certain animals such as lamas and camels can produce heavy chain only antibodies.

Each of the two heavy chains in a naturally occurring antibody comprise two identical CH3-CH2-hinge-CH1-variable regions. A dimer formed between two CH3-CH2-hinge regions provides for an Fc. Antibody production involving Fc homodimerization is primarily mediated by a large and compact interface between the two CH3 domains followed by disulfide bond formation in the hinge region (Schroeder et al. J Allergy Clin Immunol. 2010. 125 (2 Suppl 2) : S41–52; Ha et al. Front. Immunol. 2016. 7: 394) .

Compared with conventional monospecific antibodies, bispecific antibodies (BsAbs) , bind to two epitopes, which may be present on the same or different molecules. Bispecific IgG antibodies are a multimer made up of two heavy chain polypeptides and two light chain polypeptides. Expressing the different polypeptides in a cell can result in the production of different antibodies, including monospecific antibodies resulting from Fc homodimerization and bispecific antibodies resulting from Fc heterodimerization (Klein et al. MAbs. 2012.4 (6) : 653-63. ) .

A variety of different types of multispecific binding molecules can be produced using different binding domains, including antigen binding domains. The use of different CH3 regions can facilitate production of multispecific binding molecules. Publications mentioning techniques for facilitating heterodimerization mediated by CH3 regions include: Spiess et al. Molecular Immunology. 2015. 67 (2 Pt A) : 95-106; Klein et al. MAbs. 2012.4 (6) : 653-63; Carter. J Immunol Methods. 2001.248 (1-2) : 7-15; Brinkmann et al. MAbs. 2017. 9 (2) : 182-212) ; US2010/0015133, US2009/0182127, US2011/0123532, US2012/0149876, US2013/0195849, and US2021/0277150 (Zymeworks) , U.S. Patent No. 8,216,805 and Ridgway et al. Protein Eng. 1996. 9 (7) : 617-21.

SUMMARY OF INVENTION

The present invention relates to heteromultimer polypeptides comprising a heterodimeric polypeptide complex, wherein the heterodimeric polypeptide comprises two modified immunoglobulin CH3 domains. The modified immunoglobulin CH3 domains comprise substitutions favoring heterodimer formation. The different polypeptides making up the heterodimeric polypeptide complex can be combined with different binding domains to provide molecules able to bind different targets.

Different types of heteromultimer polypeptides include, for example, bispecific antibodies and different types of molecules containing two or more antibody variable regions providing for multispecificity. Additional examples include, for example, heteromultimer polypeptides comprising different types of binding agents providing for specificity, such as polypeptide receptors and ligands.

Thus, a first aspect of the present invention features a heteromultimer comprising a heterodimeric polypeptide complex, wherein the heterodimeric polypeptide complex comprises:

a) a first polypeptide comprising a first modified immunoglobulin CH3 domain having at least two amino acid substitutions relative to a wildtype immunoglobulin CH3 domain at positions selected from 349, 405 and 407, wherein the substitutions are selected from the group consisting of 349T, 349A, 349S, 349V, 349G, 405V, 405A, 405T, 405S, 405G, 407A, 407T, 407S, 407G, and 407V; and

b) a second polypeptide comprising a second modified immunoglobulin CH3 domain having at least one amino acid substitution relative to a wildtype immunoglobulin CH3 domain at positions 366 and 360, wherein the substitution is selected from the group consisting of 366Y, 366W, 366F, 366L, 360Y, 360W, 360F, and 360L, wherein numbering is according to the EU index as in Kabat.

Another aspect of the present invention is directed to a heteromultimer comprising a heterodimeric polypeptide complex, wherein the heterodimeric polypeptide complex comprises:

(a) a first modified immunoglobulin CH3 domain comprising the amino acid substitutions 405V and 407A and

(b) a second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W or 366Y; wherein numbering is according to the EU index as in Kabat

Reference to a “modified immunoglobulin CH3 domain” indicates a CH3 derived from a human Ig CH3 region and/or a polypeptide region having a sequence identity of at least 85%to SEQ ID NO: 3.

A derived sequence is produced by altering one or more amino acids from a reference sequence. Alterations can include amino acid insertions, deletions, and/or substitutions. The amino acid reference number is with respect to a reference sequence prior to any alterations. In different embodiments, the CH3 is derived from a human IgG. Corresponding changes can be made in IgM, IgA, IgD, or IgE CH3, where such changes may involve different numbering than IgG, but corresponding locations.

A wildtype immunoglobulin CH3 domain is a naturally occurring human Ig CH3 domain. In different embodiments, the wildtype Ig CH3 is a human IgG; or is IgM, IgA, IgD, and IgE.

An indicated sequence identity allows for one or more amino acid deletions, one or more additions, and/or one or more substitutions. The amino acid reference number provides the location in the corresponding wildtype sequence. In some circumstances, such as amino acid addition or deletion, the numbering from the wildtype sequence can be shifted, however the corresponding location would remain the same.

Another aspect of the present invention is directed to a heteromultimer comprising a heterodimeric polypeptide complex, wherein the dimeric polypeptide complex comprises a first polypeptide comprising a first modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, and a second polypeptide comprising a second modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, wherein:

(a) the first modified immunoglobulin CH3 domain comprises the substitution 405V, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;

(b) the first modified immunoglobulin CH3 domain comprises the substitutions 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;

(c) the first modified immunoglobulin CH3 domain comprises the substitution 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366Y;

(d) the first modified immunoglobulin CH3 domain comprises the substitution 349T, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y;

(e) the first modified immunoglobulin CH3 domain comprises the substitutions 349T and 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y;

(f) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366W;

(g) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366Y;

(h) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360W and 366Y; and

(i) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y, 366W, 435R and 436F;

wherein numbering is according to the EU index as in Kabat.

Another aspect is directed to a nucleic acid combination comprising a first nucleic acid encoding the first polypeptide of a heterodimeric complex and a second nucleic acid encoding the second polypeptide of a heterodimeric complex.

Another aspect is directed to a nucleic acid comprising a first nucleic acid encoding the first polypeptide of a heterodimeric complex and a second nucleic acid encoding the second polypeptide of a heterodimeric complex.

Another aspect is directed to a vector combination comprising a vector encoding the first polypeptide of a heterodimeric complex and a second vector encoding the second polypeptide of a heterodimeric complex.

Another aspect is directed to a host cell comprising one or more recombinant nucleic acids encoding for a heteromultimer.

Reference to “recombinant” nucleic acid indicates an association of elements different than occurring in nature. Examples of recombinant nucleic acid include heterologous regulatory elements such as a promoter and/or polyadenylation signal; the presence, association, and/or location of coding regions; and/or the of presence, association, and/or location of non-coding regions. Recombinant nucleic acid can be provided, for example, through modification of a host cell genome and/or extrachromosomal elements such as vectors.

Additional aspects include method of producing heteromultimers using a recombinant cell comprising recombinant nucleic acid encoding the multimer; and methods of obtaining heteromultimers (e.g., recovering the heteromultimers from such recombinant cells) .

Other features and advantages of the present invention are apparent from additional descriptions provided herein, including different examples and drawings. The provided examples and drawings illustrate different components and methodology useful in practicing the present invention. Such examples do not limit the claimed invention. Based on the present disclosure, the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1C. FIG. 1A illustrates a “Fab₂ × Fc” , where the Fc is homodimeric. FIG. 1B depicts a “Fab × Fc” monospecific antibody. FIG. 1C illustrates a “Fc” homodimer.

FIG. 2 shows the calculation of the percentage of correctly paired bispecific antibodies. The box around the curve indicates 3 peaks representing 3 fractions of 3 formats, which are Fab₂ × Fc, Fab × Fc, and Fc from left to right.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes modified CH3 domains comprising specific amino acid substitutions to promote heterodimer formation. The substitutions are noted with respect to IgG and/or a reference sequence. IgG subclasses (e.g., IgG₁, IgG₂, IgG₃, and IgG₄) and allotypes (e.g., for IgG1: G1m (z, a) , G1m (f) , and G1m (f, a) ) are described, for example, in Vidarsson et al. Front Immunol. 2014: 5: 520, which is herein incorporated by reference in its entirety.

Additional modifications may be also present, including modifications resulting in the same or similar activity and modifications affecting stability, half-life, and/or effector functions. Amino acid modifications that may be present in a modified CH3 domain include, but are not limited to, one or more amino acid insertions, deletions, and/or substitutions compared to the IgG CH3 domain. The modifications of the CH3 domain and the modified CH3 domains are referred to herein collectively as “CH3 modifications” , “modified CH3 domains” , “modified CH3” , “variant CH3 domains” , “CH3 variants” , “CH3 mutations” , or “mutated CH3 domains” .

Modified CH3 domains may be incorporated into molecules of choice to facilitate dimerization. In certain embodiments, the modified CH3 domains are present as a heterodimer joining two polypeptides.

Different immunoglobulin CH3 substitution combinations are identified herein favoring formation of a heterodimeric polypeptide complex, wherein the heterodimeric complex may be part of a multimer. Favoring formation of the heterodimeric complex can be useful, for example, in producing multispecific binding molecules, wherein the different polypeptides making up the heterodimeric complex have different binding domains.

Depending on the complete structure, multispecific binding molecules can have a variety of uses including diagnosis, prophylaxis, and therapeutic. Examples of therapeutic uses include cancer treatment, inflammatory diseases, autoimmunity, neurodegeneration, bleeding disorders, psoriasis, wet macular degeneration, and infections (Spiess et al. Molecular Immunology. 2015. 67 (2 Pt A) : 95-106; and Brinkmann et al. MAbs. 2017. 9 (2) : 182-212. ) .

As used herein, terms such as “heterodimers” , “heterodimeric complex” and “heterodimeric” indicate the dimer region comprises two polypeptides having a different amino acid sequence.

As used herein, terms such as “homodimers” , “homodimeric complex” and “homodimeric” indicate the dimer region comprises two polypeptides having the same amino acid sequence.

Multimers comprising a heterodimeric polypeptide complex may comprise one or more additional domains positioned outside the heterodimeric polypeptide complex, such as binding domains, antibody constant domains (or parts thereof) , and antibody hinges; and/or additional polypeptides such as light chain antibodies or binding fragments thereof. An antibody binding fragment comprises one or more variable regions.

Multimers preferably comprise two or more different binding domains. A “binding domain” refers to a polypeptide able to specifically bind to another molecule.

The terms “polypeptide, ” “protein” and “peptide” can be used interchangeably to refer to an amino acid sequence without regard to function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acid acids. Amino acids include naturally occurring amino acids and amino acids provided by cellular modification.

As used herein and in the appended claims, the singular forms “a, ” “an, ” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A, ” “B, ” “C, ” “A or B, ” “A or C, ” “B or C, ” or “A, B, or C. ”

As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or, ” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore, satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore, satisfy the requirement of the term “and/or. ”

Unless clearly indicated otherwise by the context employed the terms “or” and “and” have the same meaning as “and/or” .

Reference to terms such as “including” , “for example” , “e.g., ” “such as” followed by different members or examples, are open-ended descriptions where the listed members or examples are illustrative and other member or examples can be provided or used.

Reference to “comprise” , and variations such as “comprises” and “comprising” , used with respect to an element or group of elements is open-ended and does not exclude additional unrecited elements or method steps. Terms such as “including” , “containing” and “characterized by” are synonymous with comprising. In the different aspects and embodiments described herein reference to an open-ended term such as “comprising” can be replaced by “consisting” or “consisting essentially of” .

Reference to “consisting of” excludes any element, step, or ingredient not specified in the listed claim elements, where such element, step or ingredient is related to the claimed invention.

Reference to “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic (s) of the claimed invention.

Reference to an indicated percent identity to one or more reference sequences, and similar language throughout the specification providing for an indicated percent identity to one or more reference sequences, provides the indicated percent identity or percent identity range independently to each of the referenced sequences. In determining percent identity for a polynucleotide, RNA and the corresponding DNA are considered the same unless provided otherwise by the employed context, for example, providing reference to the polynucleotide being RNA or DNA. Corresponding RNA and DNA include uracil for thymine and replacement of the ribose backbone for the deoxyribose backbone.

Reference to a percent “identical” , “identity” and similar terminology are with respect to two sequences having maximal alignment in a particular area. The provided area is with respect to the indicated reference sequence. For example, sequence “identical” or “identity” SEQ ID NO: 1 can be calculated by determining the number of identical amino acids in aligned sequences, dividing by the total number of amino acids in SEQ ID NO: 1 (125 amino acids) and multiplying by 100. Percent “identical” or “identity” for nucleic acid sequences can be determined in an analogous manner where nucleotides to the reference sequence are aligned to achieve maximal alignment taking into account nucleotide differences and gaps, dividing by the total number of nucleotides in the reference sequence and multiplying by 100.

Throughout the application, unless clearly indicated otherwise by the context, when different variable components are provided, any variable of one component may be used with any variable on another component. For example, in the case where SEQ ID NO: X and SEQ ID NO: Y refer to two different sequences, reference to a first modified IgG CH3 domain with a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO: X in combination with a second modified immunoglobulin CH3 with a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO: Y, include embodiments where the first modified immunoglobulin CH3 domain can have a sequence identity of at least 95%to SEQ ID NO: X, and the second modified immunoglobulin CH3 domain can have a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO: Y; the first modified immunoglobulin CH3 domain can have a sequence identity of at least 96%to SEQ ID NO: X, and the second modified immunoglobulin CH3 domain has a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO: Y; the first modified immunoglobulin CH3 domain can have a sequence identity of at least 97%to SEQ ID NO: X, and the second modified immunoglobulin CH3 domain has a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO: Y; the first modified immunoglobulin CH3 domain can have a sequence identity of at least 98%to SEQ ID NO: X, and the second modified immunoglobulin CH3 domain has a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO: Y; and so forth.

The term “about” refers to a value within 10%of the underlying parameter (i.e., plus or minus 10%) . For example, “about 1: 10” includes 1.1: 10.1 or 0.9: 9.9, and “about 5 hours” includes 4.5 hours or 5.5 hours. The term “about” at the beginning of a string of values modifies each of the values by 10%. In certain embodiments, about refers to plus or minus 5%of an indicated value.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood to one of ordinary skill in the art to which this invention pertains.

The definitions provided herein, including those in the present section and other sections of the application, apply throughout the present application.

The description has been separated into various sections and paragraphs, and provides examples of various embodiments. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiment. The provided descriptions have broad application and encompass all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest the scope of the disclosure, including the claims (unless otherwise provided in the clams) , is limited to these examples.

The instant invention is generally disclosed herein using affirmative language to describe the numerous embodiments of the instant invention. The instant invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments of the instant invention, materials and/or method steps are excluded. Thus, even though the instant invention is generally not expressed herein in terms of what the instant invention does not include, embodiments that are not expressly excluded in the instant invention are nevertheless disclosed herein.

Various references including articles and patent publications are cited or described in the background and throughout the specification. Each of these references is herein incorporated by reference in their entirety. None of the references are admitted to be prior art with respect to any inventions disclosed or claimed. In some cases, particular references are indicated to be incorporated by reference herein to highlight the incorporation.

I. Heterodimeric Polypeptide Complex

The present invention provides for CH3 substitution combinations on different polypeptides favoring formation of a heterodimeric polypeptide complex. Reference to “substitution” , “mutation” and “modification” of an amino acid provides for a different amino acid than in the wildtype sequence, and is not a description concerning production.

Heterodimer formation can be favored in different ways such as knobs-into-holes (KIH or knob-in-hole) , electrostatic, and/or cysteine bond formation (Wei et al. Oncotarget. 2017. 8 (31) : 51037-51049; Klein et al. MAbs. 2012.4 (6) : 653-63; and Brinkmann et al. MAbs. 2017. 9 (2) : 182-212) . Without being limited to any theory, one or more amino acids in the provided amino acid combinations of the first and second polypeptide making up the heterodimer complex may favor heterodimerization through a knob-in-hole mechanism.

In certain embodiments, the heterodimeric polypeptide complex comprises:

In certain embodiments, the dimeric polypeptide complex comprises a first polypeptide comprising a first modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, and a second polypeptide comprising a second modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, wherein:

(a) the first modified immunoglobulin CH3 domain comprises the substitution 405V such as F405V, and the second modified immunoglobulin CH3 domain comprises the substitution 366W such as T366W;

(b) the first modified immunoglobulin CH3 domain comprises the substitutions 405V and 407A such as F405V and F407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366W such as T366W;

(c) the first modified immunoglobulin CH3 domain comprises the substitution 407A such as Y407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366Y such as T66Y;

(d) the first modified immunoglobulin CH3 domain comprises the substitution 349T such as Y349T, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y such as K360Y;

(e) the first modified immunoglobulin CH3 domain comprises the substitutions 349T and 407A such as Y349T and Y407A, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y such as K360Y;

(f) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A such as Y349T, F405V, and Y407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366W such a K360Y and T366W;

(g) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A such as Y349T, F405V, and Y407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366Y such as K360Y and T366Y;

(h) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A such as Y349T, F405V, and Y407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360W and 366Y such as K360W and T366Y; and

(i) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A such as Y349T, F405V, and Y407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y, 366W, 435R and 436F such as K360Y, T366W, H435R, and Y436F;

wherein numbering is according to the EU index as in Kabat.

In certain embodiments, reference to a modified immunoglobulin CH3 domain indicates a CH3 derived from a human Ig CH3 region by providing for the indicated substitutions and up to 10 additional modifications. In further embodiments 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 amino acids modification are provided. In further embodiments the alterations are with respect to human IgG, IgM, IgA, IgD, and IgE; human IgG; human IgG₁; or human IgG₄.

Human immunoglobulins can be assigned to five major isotypes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG₁, IgG₂, IgG₃ and IgG₄. Antibody light chains of vertebrate species may be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ) , based on the amino acid sequences of their constant domains. General principles of antibody molecule structure and various techniques relevant to the production of antibodies are provided in, e.g., Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (1988) .

For all positions discussed in the present application, numbering of an immunoglobulin heavy chain is according to the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda) . The “EU index as in Kabat” refers to the residue numbering of the human IgG₁ EU antibody.

Different locations for modifications are described herein referencing IgG numbering. Corresponding changes can be made in IgM, IgA, IgD, or IgE, where such changes may involve different numbering than IgG, but corresponding locations. (See, for example, Davis et al. Protein Engineering, Design and Selection. 2010. 23 (4) : 195-202 and Sun et al. J Mol Biol. 2005. 353 (1) : 155-73, both of which are incorporated by reference herein in their entirety) .

Reference to amino acid modification indicates a deletion, substitution or addition. When more than one amino acid modification is present, each modification can independently be either a deletion, substitution or addition.

In certain embodiments, reference to a modified immunoglobulin CH3 domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG₁ CH3. In further embodiments the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 3; or in addition to the indicated substitutions can differ from SEQ ID NO: 3 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

In certain embodiments, reference to a modified immunoglobulin CH3 domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG₂ CH3. In further embodiments the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 19; or in addition to the indicated substitutions can differ from SEQ ID NO: 19 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

In certain embodiments, reference to a modified immunoglobulin CH3 domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG₃ CH3. In further embodiments the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 20; or in addition to the indicated substitutions can differ from SEQ ID NO: 20 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

In certain embodiments, reference to a modified immunoglobulin CH3 domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG₄ CH3. In further embodiments the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 21; or in addition to the indicated substitutions can differ from SEQ ID NO: 21 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

In certain embodiments, additional modifications that can be present include, for example, modifications favoring heterodimer formation, modifications altering one or more Fc effector activity, and/or modifications not significantly decreasing (e.g., less than 20%or less than 10%impact) heterodimer formation.

In certain embodiments, the first polypeptide further comprises a first CH2 immunoglobulin domain joined to the amino terminus of the first CH3 immunoglobulin domain and the second polypeptide further comprises a second CH2 immunoglobulin domain joined to the amino terminus of the second CH3 amino terminus. The first and second CH2 immunoglobulin domains may be the same or different.

In certain embodiments, the first CH3-CH2 hinge domain and the second CH3-CH2 hinge domain are derived from a human Ig CH3-CH2 region by, in addition to providing for the indicated substitutions, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modification are provided or any subcombination thereof, for example, 0 to 15, 0 to 10, 0 to 5, or 0 to 3 additional amino acids are modified. In further embodiments the modifications are with respect to any of human IgG, IgM, IgA, IgD, and IgE; human IgG; or human IgG₁ or IgG₄.

Preferably, the first polypeptide further comprises a first immunoglobulin hinge region joined to the amino terminus of the first CH2 domain providing a first CH3-CH2-hinge domain and the second polypeptide further comprises a second CH2 immunoglobulin hinge region joined to the amino terminus of the second CH2 amino terminus providing a second CH3-CH2-hinge domain, wherein the hinge domains are joined by one or more disulfide bonds. The heterodimeric complex formed between the first and the second CH3-CH2-hinge domain corresponds to an Fc region.

The Fc may also provide for different effector functions such as antibody-dependent cellular cytotoxicity (ADCC) , antibody-dependent cellular phagocytosis (ADCP) , and complement-dependent cytotoxicity (CDC) .

Additional possible advantages include potential for increased half-life (e.g., binding to FcRn) and purification (e.g., using protein A or protein G affinity columns) .

In certain embodiments, the first CH3-CH2-hinge domain and the second CH3-CH2 hinge domain are derived from a human Ig CH3-CH2-hinge domain by, in addition to providing for the indicated substitutions, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or any subcombination thereof, of example 0-15, 0 to 10, 0 to 5, 0-3 amino acids are modified. In further embodiments the modifications are with respect to any of human IgG, IgM, IgA, IgD, and IgE; human IgG; or human IgG₁ or IgG₄.

In certain embodiments, reference to a modified immunoglobulin CH3-CH2-hinge domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to a IgG₁ CH3-CH2-hinge domain. In further embodiments the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 12; or in addition to the indicated substitutions can differ from SEQ ID NO: 12 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

IgG₂, IgG₃ and IgG₄ CH2 domains are highly homologous with IgG₁ CH2. SEQ ID NOs: 22, 23 and 24 provide examples of CH3-CH2-hinge domains from IgG₂, IgG₃ and IgG₄.

In certain embodiments, reference to a modified immunoglobulin CH3-CH2-hinge domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG₂ CH3. In further embodiments the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 22; or in addition to the indicated substitutions can differ from SEQ ID NO: 22 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

In certain embodiments, reference to a modified immunoglobulin CH3-CH2-hinge domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG₃ CH3. In further embodiments the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 23; or in addition to the indicated substitutions can differ from SEQ ID NO: 23 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

In certain embodiments, reference to a modified immunoglobulin CH3-CH2-hinge domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG₄ CH3. In further embodiments the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 24; or in addition to the indicated substitutions can differ from SEQ ID NO: 24 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

In some embodiments, the heterodimeric polypeptide may comprise not only, substitutions described herein facilitating heterodimer formation, but also comprise one or more Fc modifications increasing stability, increasing half-life, and/or altering effector functions.

Examples of Fc modifications increasing cellular cytotoxic effector functions include:

i. S239D and I332E ( “DE” ) ;

ii. S239D, I332E, and A330L ( “DLE” ) ;

iii. S239D; I332E; S298A, E333A, and K334A;

iv. G236A, A330L, and I332E ( “GAALIE” ) ;

v. G236A, S239D, A330L, and I332E ( “GASDALIE” ) ;

vi. F243L, R292P, Y300L, V305I, and P396L ( “LPLIL” ) ;

vii. M252Y, S254T, and T256E ( “YTE” ) ; and

viii. T307A, E380A, and N434A ( “AAA” )

(U.S. Pat. No. 10,184,000; Liu et al. Antibodies (Basel) . 2020. 9 (4) : 64; Lazar et al. PNAS. 2006. 103 (11) : 4005-4010; each of which is herein incorporated by reference in its entirety) .

The Fc can prolong half-life in circulation through its interaction with FcRn. Half-life extending modifications include:

i. M428L and N434S ( “LS” ) ;

ii. M252Y and T256D ( “YD” ) ;

iii. T256D and T307Q ( “DQ” ) ; and

iv. T256D and T307W ( “DW” ) .

In some embodiments, the heterodimeric polypeptide may comprise Fc modifications that abolish or reduce immune effector functions. Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (FcγR) can be useful limiting unwanted inflammatory responses to therapeutic antibodies. Fc variants that abolish or reduce immune effector functions include:

i. P329G, L234A, and L235A ( “LALA-PG” ) ; and

ii. L234A and L235A ( “LALA” )

(Wilkinson et al. PLOS ONE. 2021.16 (12) : e0260954, herein incorporated by reference in its entirety) .

Antibodies like all polypeptides have an isoelectric point (pI) , which is generally defined as the pH at which a polypeptide carries no net charge. Protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pI) of the protein. It is possible to optimize solubility by modifying the number and location of ionizable residues in the antibody to adjust the pI (see U.S. Patent No. 10,457,742, which is herein incorporated by reference in its entirety) .

In certain embodiments, the first and second polypeptide comprise CH3 modifications provided for in Table 1:

Table 1

In certain embodiments, heteromultimers are produced (e.g., in a cell) , or provided in preparation, wherein of the total multimers, the heteromultimers are about 90%or greater, about 93%or greater, about 95%or greater, or about 97%or greater of the total multimers. Percentage of heteromultimers can be assessed as provided in the Examples below (e.g., CE-SDS or HPLC) .

II. Binding Domains

Different binding domains can be provided to different polypeptide making up a heteromultimer. In certain embodiments, a particular binding domain can specifically bind to another molecule. Reference to “specifically” or “specific” with respect to binding does not require absolute specificity. Rather the specificity is provided by the binding domain able to distinguish the targeted molecule from most or all other molecule that are present. In certain embodiments, the binding domain is able to distinguish the targeted molecule from at least 75%, 85%, 95, or 99%from other molecules present in a biological sample or subject containing the targeted molecules. Biological molecules containing targeted molecules, may include, for example, material obtained from an animal (e.g., human) and/or present in an animal (e.g., human) .

The different polypeptides making up the heterodimeric polypeptide complex can be combined with different binding domains to provide for molecules that are able to bind different targets. Different types of heteromultimer polypeptides can be produced, including, for example, bispecific antibodies and different types of molecules containing two or more antibody variable regions, providing for multispecificity, polypeptide receptors, and ligands.

II. A. Antigen Binding Domains

In certain embodiments, the binding domain is an antigen binding domain. An “antigen binding domain” refers to one or more immunoglobulin variable regions able to bind to an epitope present on an antigen. An immunoglobulin variable region contains three complementary determining regions (CDRs) interspaced within a framework (FR) . Different types and sources of immunoglobulin variable regions can be used, such as variable regions obtained from primate (e.g., humans, camels, monkeys) antibodies and variable regions obtained from other animals such as sharks, camels, llamas, mice, and rats. In certain embodiments, framework regions for human immunoglobulins use humanized or human framework regions.

Antigen binding domains comprise at least one variable region. In certain embodiments, two variable regions are provided, for example, domains provided by a heavy and a light chain variable region contribute to binding. In naturally occurring human antibodies, the heavy chain can be viewed as an extension from the CH3 domain, while the light chain variable region provides an additional polypeptide. Certain animals provide for naturally occurring antibodies with only a heavy chain, for example, camel, shark, and llama single domain heavy chain antibodies (see Flajnik et al. PLoS Biol. 2011. 9 (8) : e1001120, which is herein incorporated by reference in its entirety) .

In certain embodiments, the heteromultimer contains two or more different antigen binding domains. Each antigen binding domain can be a single domain binding site provided by one variable region or a two-domain binding site provided by two immunoglobulin variable regions.

An antigen binding domain can be provided for by a variety of different antigen binding fragments comprising such a domain. Examples of antibody binding fragments include VH, VL, VHH (sdAb or dAb) , Fab, Fab’, F (ab’) ₂, scFab, scFv, (scFv) ₂, Fv, taFv, DART and (bispecific T-cell engager) .

Multispecificity can be provided for by a variety of different formats (see Spiess et al. Molecular Immunology. 2015. 67 (2 Pt A) : 95-106 and Brinkmann et al. MAbs. 2017. 9 (2) : 182-212, both of which are incorporated by reference herein in their entirety) . Examples of such formats include scFv-CH3, scFv-Fc, scFab (single chain Fab) , Fab-scFab, orthogonal Fab, DuetMab, CrossMAb, Ig-Fv, scFab-Fc-scFv2, scFab-Fc-scFv, DVD-Ig^TM (dual variable domain immunoglobulin) , taFv (tandem scFv) -Fc, scFv-Fc-Fv, Fab-Fc-scFv, Fab-scFv-Fc, DART-Fc, scFv-CH3, and TriFabs.

Reference to “DuetMab” refers to the replacement of the native interchain disulfide bond with one of the 2 CH1-CL interfaces with an engineered interchain disulfide bond using knobs-into-holes technology (see Mazor et al. MAbs. 2015. 7 (2) : 377-389) .

Reference to “CrossMAb” is a type of knobs-into-holes technology in which either the light chain of one Fab arm is exchanged by the Fd of the corresponding heavy chain, or only one pair of the variable or constant domain of one Fab is swapped between the light and heavy chain (see Brinkmann et al. MAbs. 2017. 9 (2) : 182-212) .

Reference to “DART” indicates dual-affinity retargeting proteins (see Brinkmann et al. MAbs. 2017. 9 (2) : 182-212) .

Reference toindicates a fusion protein that forms a bridge between cancer cells and cytotoxic T-cells. Canonicalmolecules are comprised of two scFvs from an anti-TAA (tumor-associated antigen) and an anti-CD3 monoclonal antibody with a short linker connecting them in tandem (see Zhou et al. Biomarker Research. 2021. 9 (38) : hyperlink/doi. org/10.1186/s40364-021-00294-9) .

Reference to “TriFabs” indicate two regular Fabs fused through flexible 20-residue peptide linkers, (G₄S) ₄, to an asymmetric Fab-like entity as heterodimerization module or stem region. The overall structure of TriFabs resembles that of an IgG with the Fc region substituted by the stem region (see Brinkmann et al. MAbs. 2017. 9 (2) : 182-212) .

Reference to “DVD-Ig^TM” indicates a tetra-specific antibody format (four-in-one) that is constructed by fusing two dual variable domain immunoglobulin antibodies (see DiGiammarino et al. Methods Mol Biol. 2012. 899: 145-56, which is herein incorporated by reference in its entirety) .

Reference to “antibody” includes both single domain antibodies containing variable regions provided by an antibody heavy chain (e.g., VHH) and two domain antibodies containing variable regions provided by antibody heavy and light chains. Antibodies can be provided in different preparations, including polyclonal antibodies or monoclonal antibodies. Monoclonal antibodies refer to a population of antibodies having substantially the same structure, where any structural variation is provided by a hybridoma expressing the antibody or cell lines designed to express only a particular antibody (e.g., recombinant cell comprising antibody encoding nucleic acid) .

The base subunit for a full-length human antibody is two heavy chains and two light chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region and a heavy chain constant region (comprised of at least domains CH1, hinge, CH2 and CH3) . Each light chain is comprised of a light chain variable region and a light chain constant region. The VH and the VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions, interspersed with framework regions. Each VH and VL is composed of three complementarity determining regions and four framework segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

As used herein, the term “multispecific antibody” refers to an antibody that comprises multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein) . In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or do not substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein) . In an embodiment, a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, a multispecific antibody is a bispecific antibody molecule, a trispecific antibody, or a tetraspecific antibody molecule.

In some embodiments, antigen binding domains can be from any animal origin including birds and mammals (e.g., human, primate, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken) . In preferred embodiments, the antibody variable regions are of human origin.

In certain embodiments, the antigen binding domains are selected from the group consisting of CD19 (B4, CVID3, B-lymphocyte antigen CD19) , CD20 (MS41, Bp35, B-lymphocyte antigen CD20) , EpCAM (epithelial cell adhesion molecule, CD326, GA733-2, M1S2, M4S1, MIC18, TACSTD1, TROP1) , CEA (Carcinoembryonic antigen-related cell adhesion molecule) , PSMA (Prostate-specific membrane antigen, FOLH1, NAALAD1, PSM, GIG27) , GD2 Ganglioside, CD30 (TNFRSF8, Tumor necrosis factor receptor superfamily member 8) , CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1, T10) , CD47 (Leukocyte surface antigen CD47, MER6) , CD52 (CAMPATH-1 antigen, CDW52, HE5) , c-Met (DA11, HGFR, AUTS9, RCCP2, DFNB97) , CA19-9 (ST6GALNAC6, Alpha-N-acetylgalactosaminide alpha-2, 6-sialyltransferase 6, SIAT7F) , CA72-4 (cancer antigen 72-4) , CAM 17.1, CD1a (R4, T6, CD1, FCB6, HTA1) , CD5 (T1, LEU1) , CD11A (integrin subunit alpha L, ITGAL, LFA-1, LFA1a) , CD40 (p50, Bp50, CDW40, TNFRSF5) , CD44 (CDW44, CSPG8, ECM-III, ECMR-III, H-CAM, HCELL, HUTCH-1, HUTCH-I, Hermes-1, IN, LHR, MC56, MDU2, MDU3, MIC4, Pgp1) , B7-H3 (CD276, B7RP-2, 4Ig-B7-H3) , B7-H4 (B7X, B7S1, VTCN1, V-set domain containing T cell activation inhibitor 1, PRO1291) , B7-H6 (NCR3LG1, natural killer cell cytotoxicity receptor 3 ligand 1, DKFZp686O24166) , and PD-L (CD274, B7-H, hPD-L1, PDCD1L1, PDCD1LG1, B7H1) .

As used herein, the term “human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.

As used herein, the term “antigen binding fragment” refers to one or more polypeptides comprising an immunoglobulin variable region (e.g., single and two domain binding regions) such as, a diabody, a Fab, a Fab′, a F (ab′) ₂, scFab, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) ₂, a bispecific dsFv (dsFv-dsFv′) , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , a single domain antibody (sdAb) an scFv dimer (bivalent diabody) , taFv, a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody or a bivalent domain antibody. In certain embodiments, the antigen binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the heavy chain. The Fd segment is approximately the first 220 amino acids from the N-terminus of the heavy chain, contained within the heavy chain of the Fab. According to other particular embodiments, the antigen binding fragment comprises Fab and F (ab′) .

As used herein, the term “single-domain antibody” (sdAb, dAb or VHH) refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.

As used herein, the term “Fab” or “Fab fragment” refer to an antibody fragment composed of VH, CH1, VL and CL domains.

As used herein, the term "Fv" refers to the minimum antibody fragment that contains a complete antigen-recognition and antigen binding site. This region may be made of a heavy chain variable region or a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association.

As used herein, the term, “Single-chain Fv, ” “single-chain antibody, ” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Ashmad et al. Clin Dev Immunol. 2012.2102: 980250, which is herein incorporated by reference in its entirety.

In certain embodiments, specific binding refers to preferential binding of the two proteins with typically an equilibrium dissociation constant (K_D) of about 1×10^-8 M or less, for example about 1×10^-9 M or less, about 1×10^-10 M or less, about 1×10^-11 M or less, or about 1×10^-12 M or less, typically with the K_D that is at least one hundred-fold less than its K_D for binding to a non-specific antigen (e.g., BSA, casein) .

As used herein, the term "antibody derivative" refers to a molecule comprising a full-length antibody or an antigen binding fragment thereof, wherein one or more amino acids are chemically modified or substituted. Chemical modifications that can be used in antibody derivatives include, e.g., alkylation, PEGylation, acylation, ester formation or amide formation or the like, e.g., for linking the antibody to a second molecule. Exemplary modifications include PEGylation (e.g., cysteine-PEGylation) , biotinylation, radiolabeling, and conjugation with a second agent (such as a cytotoxic agent) .

II. B. Additional Binding Domains

Additional binding domains include a receptor able to bind to a ligand, and a ligand able to bind to a receptor. Fusion proteins providing for specific binding can be produced by appending a receptor or ligand onto one or both polypeptides making a heterodimeric immunoglobulin complex comprising a CH3 dimer. Multispecificity can be provided using different formats such as appending different receptors to each polypeptide; appending different ligands to each polypeptide; appending a receptor to one polypeptide and a ligand to another polypeptide; and attaching a receptor or ligand to one polypeptide and providing an antigen domain to another polypeptide. Ligand and receptor combinations present on a particular multimer are selected to not bind to each other.

Different degrees of specificity and configurations can be provided through use of receptors, ligands, and/or antigen binding domains. Configurations such as those described in II. A. (Antigen Binding Domains) supra can be employed, wherein one or more antigen binding domains is replaced by a receptor or ligand.

In different embodiments, one or more antigen binding domains are provided as described in II. A. supra.

In certain embodiments, the heteromultimer comprises a heterodimeric immunoglobulin complex comprising a CH3-CH2 heterodimer as provided for in II. A. supra.

In preferred embodiments, the heterodimeric immunoglobulin complex comprises a Fc as provided for example in II. A. supra.

In certain embodiments, the heterodimeric multimer is an immunoadhesin or an antibody-immunoadhesin chimera. An immunoadhesin comprises an Fc region and receptor binding domains made up of receptors and/or ligands. An antibody-immunoadhesin chimera comprises an Fc region at least one binding domain joined to a receptor or ligand and at least one binding domain joined to an antigen binding domain (e.g., antigen binding fragment) . Examples of immunoadhesins and antibody-immunoadhesins chimeras are described, for example, in Pérez et al. Recent Papers on Anti-Infective Drug Discovery. 2009. 4 (3) : 183-189, and U.S. Patent No 8,216,805, both of which are incorporated by reference herein in their entirety.

III. Methods of preparing heterodimeric polypeptides

Heteromultimers comprising a heterodimeric polypeptide complex can be produced by expressing the individual polypeptides and allowing for the formation of the heterodimeric polypeptide complex. The multimers may also comprise additional polypeptides, for example, antibody light chains or binding fragments thereof. The produced heteromultimer can be purified from other material, including homodimers as described in the Examples below or use alternative purification techniques. (see U.S. Patent No. 10,875,931 and Yao et al. Nature Communications. 2022. 13: 1539; each of which is herein incorporated by reference in its entirety. )

Polypeptides can be produced from a single nucleic acid encoding for each of the polypeptides or through the use of multiple nucleic acids encoding for the different polypeptides. The different nucleic acids can exist inside a host cell as extrachromosomal elements and/or be integrated into a host genome. In addition to sequences encoding for polypeptides comprising heterodimers, encoding sequences can be provided for additional polypeptides associated with heterodimers, for example, in the case of an antibody, antibody light chains, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies.

In certain embodiments, the heteromultimer is produced in a host cell comprising a first recombinant nucleic acid segment encoding for a first polypeptide of the heterodimer and a second recombinant nucleic acid segment encoding for a second polypeptide. The first and second recombinant nucleic acid segments can be provided on the same or on different nucleic acids. Reference to “recombinant nucleic acid segment” refers to a combination of different nucleic acids than those found in nature. For example, the recombinant nucleic acid segment can exist in a vector along with vector elements and/or be integrated inside a host genome.

In certain embodiments, nucleic acid encoding the first and second polypeptides are provided on one or more nucleic acid vectors. Vectors can be produced by different methods including, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. In certain embodiments, the vector comprises a nucleotide sequence encoding a heterodimeric polypeptide, operably linked to a promoter.

“Vector” refers to an extrachromosomal polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, a polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, virus, animal, plant, and reconstituted biological systems.

“Host cell” refers to a cell able to express nucleic acid encoding for multimer polypeptides. In certain embodiments, the host cell is bacterial host; a mammalian cell line, such as a human cell line (e.g., HeLa) ; a hamster cell line (e.g., CHO) ; or a murine cell line. An expression vector can be used to facilitate delivery and expression of an encoding nucleic acid to a host cell. The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a heterodimeric polypeptide of the invention. A variety of host-expression vector systems may be utilized to express the heterodimeric polypeptides described herein (e.g., antibodies) (see, e.g., U.S. Pat. No. 5,807,715, which is herein incorporated by reference in its entirety) .

Heterodimeric polypeptides (e.g., antibodies) may be purified by different methods such as chromatography (e.g., ion exchange, affinity, Protein A, and/or sizing column chromatography) , centrifugation, and differential solubility.

Polypeptides expressed with a signal sequence can generally be recovered from the culture medium as a secreted polypeptide, while polypeptides expressed without a signal sequence may be recovered from host cell lysate. If the heterodimeric polypeptide is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton^TM X-100) .

Expression of heteromultimers can be facilitated using signal peptides (see, for example, Haryadi et al. PLOS ONE. 2015. 10 (2) : e0116878. doi: 10.1371/journal, describing optimization of heavy chain and light chain signal peptides for increase expression, which is herein incorporated by reference in its entirety) .

Multimers can be purified, for example, by methods including ion-exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, size-exclusion chromatography, dialysis, or affinity chromatography (see U.S. Patent No. 10,457, 742, which is herein incorporated by reference in its entirety) .

IV. Embodiments

The application includes, but is not limited to, the following numbered embodiments:

Embodiment 1. A heteromultimer comprising a heterodimeric polypeptide complex, wherein the heterodimeric polypeptide complex comprises:

Embodiment 2. The heteromultimer of Embodiment 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 405 and 407; and the second modified immunoglobulin CH3 domain comprises a substitution at position 366.

Embodiment 3. The heteromultimer of Embodiment 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 349 and 407; and the second modified immunoglobulin CH3 domain comprises a substitution at position 360.

Embodiment 4. The heteromultimer of Embodiment 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 349, 405 and 407; and the second modified immunoglobulin CH3 domain comprises substitutions at positions 360 and 366.

Embodiment 5. The heteromultimer of any one of Embodiments 1-4, wherein the substitutions are selected from 349T, 405V, 407A, 366W, 366Y, 360W, and 360Y.

Embodiment 6. A heteromultimer comprising a heterodimeric polypeptide complex, wherein the dimeric polypeptide complex comprises a first polypeptide comprising a first modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, and a second polypeptide comprising a second modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, wherein:

(a) the first modified immunoglobulin CH3 domain comprises the substitutions 405V, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;

(h) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360W and 366Y; or

wherein numbering is according to the EU index as in Kabat.

Embodiment 7. The heteromultimer of Embodiment 1, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitutions 405V and 407A and the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W or 366Y.

Embodiment 8. The heteromultimer of Embodiment 7, wherein the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W.

Embodiment 9. The heteromultimer of Embodiment 8, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 5; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 6.

Embodiment 10. The heteromultimer of Embodiment 8, wherein the second modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 360Y.

Embodiment 11. The heteromultimer of Embodiment 10, where in the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T.

Embodiment 12. The heteromultimer of Embodiment 10, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 8.

Embodiment 13. The heteromultimer of Embodiment 11, wherein the second modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitutions 435R and 436F.

Embodiment 14. The heteromultimer of Embodiment 13, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 11.

Embodiment 15. The heteromultimer of Embodiment 7, wherein the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T and second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitutions 366Y and 360Y.

Embodiment 16. The heteromultimer of Embodiment 15, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 9.

Embodiment 17. The heteromultimer of Embodiment 7, wherein the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T and the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitutions 366Y and 360W.

Embodiment 18. The heteromultimer of Embodiment 17, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 10.

Embodiment 19. The heteromultimer of Embodiment 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitution 349T and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 360Y.

Embodiment 20. The heteromultimer of Embodiment 19, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 13; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 14.

Embodiment 21. The heteromultimer of Embodiment 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitutions 349T and 405V and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 360Y.

Embodiment 22. The heteromultimer of Embodiment 21, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 15; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 14.

Embodiment 23. The heteromultimer of Embodiment 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitutions 349T and 407A and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 360Y.

Embodiment 24. The heteromultimer of Embodiment 23, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 16; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 14.

Embodiment 25. The heteromultimer of Embodiment 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitution 405V and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 366W.

Embodiment 26. The heteromultimer of Embodiment 25, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 17; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 6.

Embodiment 27. The heteromultimer of any one of Embodiments 1-26, wherein the first modified immunoglobulin CH3 domain and the second first modified immunoglobulin CH3 domain are derived from a human IgG.

Embodiment 27a. The heteromultimer of Embodiment 27, wherein the human IgG is selected from IgG₁, IgG₂, IgG₃, or IgG₄.

Embodiment 27b. The heteromultimer of Embodiment 27a, wherein the human IgG is most preferably IgG₁.

Embodiment 28. The heteromultimer of any one of Embodiments 1-27a, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least99%with SEQ ID NO: 3 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 3.

Embodiment 29. The heteromultimer of any one of Embodiments 1-27a, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 19 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 19.

Embodiment 30. The heteromultimer of any one of Embodiments 1-27a, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 20 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 20.

Embodiment 31. The heteromultimer of any one of Embodiments 1-27a, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 21 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 21.

Embodiment 32. The heteromultimer of any one of Embodiments 1-27a, wherein the first polypeptide further comprises a first CH2 immunoglobulin domain joined to the amino terminus of the first CH3 immunoglobulin domain and the second polypeptide further comprises a second CH2 immunoglobulin domain joined to the amino terminus of the second CH3 amino terminus, wherein the first and second CH2 immunoglobulin domains may be the same or different.

Embodiment 33. The heteromultimer of Embodiment 32, wherein the first CH3-CH2-hinge domain is an IgG₁ or IgG₄ and the second CH3-CH2-hinge domain is an IgG₁ or IgG₄.

Embodiment 34. The heteromultimer of any one of Embodiments 1-33, wherein the first polypeptide further comprises a first immunoglobulin hinge region joined to the amino terminus of the first CH2 domain providing a first CH3-CH2-hinge domain and the second polypeptide further comprises a second immunoglobulin hinge region joined to the amino terminus of the second CH2 amino terminus providing a second CH3-CH2-hinge domain, wherein the first and second hinge regions are joined by one or more disulfide bonds, and wherein the first CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at least 90%to SEQ ID NOs: 12, 22, 23 or 24 and the second CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at 85%to SEQ ID NOs: 12, 22, 23 or 24.

Embodiment 35. The heteromultimer of Embodiment 34, wherein the first CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or at least 99%to any of SEQ ID NO: 12, 22, 23, or 24 and the second CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or at least 99%to SEQ ID NO: 12, 22, 23, or 24; or in addition to the indicated substitutions can differ from any of SEQ ID NO: 12, 22, 23, or 24 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.

Embodiment 36. The heteromultimer of Embodiments 1 or 6, wherein either the first polypeptide comprises the sequence of SEQ ID NO: 4 and the second polypeptide comprises the sequence of SEQ ID NO: 6; the first polypeptide comprises the sequence of SEQ ID NO: 5 and the second polypeptide comprises the sequence of SEQ ID NO: 6; the first polypeptide comprises the sequence of SEQ ID NO: 13 and the second polypeptide comprises the sequence of SEQ ID NO: 14; the first polypeptide comprises the sequence of SEQ ID NO: 15 and the second polypeptide comprises the sequence of SEQ ID NO: 14; the first polypeptide comprises the sequence of SEQ ID NO: 16 and the second polypeptide comprises the sequence of SEQ ID NO: 14; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 8; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 9; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 10; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 11; or the first polypeptide comprises the sequence of SEQ ID NO: 17 and the second polypeptide comprises the sequence of SEQ ID NO: 6.

Embodiment 37. The heteromultimer of any one of Embodiments 1-36, wherein the first polypeptide further comprises a first binding domain and the second polypeptide further comprises a second binding domain, wherein the first and second binding domains are different from each other.

Embodiment 38. The heteromultimer of Embodiment 37, wherein the first and second binding domains are independently selected from a receptor or a ligand.

Embodiment 39. The heteromultimer of Embodiment 37, wherein the first binding domain is a first antigen binding domain and the second binding domain is a second antigen binding domain, wherein the first and second antigen binding domains bind to different epitopes.

Embodiment 40. The heteromultimer of Embodiment 39, wherein the first and second antigen binding domains bind to different antigens.

Embodiment 41. The heteromultimer of any one of Embodiments 39 or 40, wherein at least one of the first and second polypeptides further comprises a third antigen binding domain such as a third antigen binding domain comprising a third Fab, a third scFv, or a third VHH.

Embodiment 42. The heteromultimer of Embodiment 41, wherein the first polypeptide further comprises a third antigen binding domain, and the second polypeptide further comprises a fourth antigen binding domain.

Embodiment 43. The heteromultimer of Embodiment 42, wherein the first polypeptide further comprises a fourth antigen binding domain, such as a fourth antigen binding domain comprising a fourth Fab, a fourth scFv, or a fourth VHH.

Embodiment 44. The heteromultimer of any one of claims 39-43, wherein each antigen binding domain is selected from the group consisting of a VH, VL, VHH (sdAb or dAb) , Fab, Fab’, F (ab’) ₂, scFab, scFv, (scFv) ₂, Fv, taFv, DART and bispecific T-cell engager.

Embodiment 45. The heteromultimer of Embodiments 34 or 35, wherein the heteromultimer is selected from the group comprising a DART-Fc, CrossMAb, DuetMab, TriFabs, (controlled Fab arm exchange generated bispecific antibodies) , scFv-CH3, scFv-Fc, scFab (single chain Fab) , Fab-scFab, orthogonal Fab, DuetMab, CrossMAb, Ig-Fv, scFab-Fc-scFv2, scFab-Fc-scFv, DVD-Ig^TM (dual variable domain immunoglobulin) , taFv (tandem scFv) -Fc, scFv-Fc-Fv, Fab-Fc-scFv, Fab-scFv-Fc, and scFv-CH3.

Embodiment 46. The heteromultimer of any one of Embodiments 39-45, wherein the heteromultimer binds to one, two, three, four or more antigenic polypeptides.

Embodiment 47. The heteromultimer of Embodiment 46, wherein the antigenic polypeptides are selected from the group consisting of CD19, CD20, EpCAM, CEA, PSMA, GD2, CD30, CD38, CD47, CD52, c-Met, CA19-9, CA72-4, CAM 17.1, CD1a, CD5, CD11A, CD40, CD44, B7-H3, B7-H4, B7-H6, PD-L1 or antigenic fragments thereof.

Embodiment 48. The heteromultimer of any one of Embodiments 1-35 or 37-47, wherein the first and/or second polypeptide further comprises one or more Fc mutations selected from the group comprising:

i. S239D and I332E ( “DE” ) ;

ii. S239D, I332E, and A330L ( “DLE” ) ;

iii. S239D;

iv. I332E;

v. S298A, E333A, and K334A;

vi. G236A, A330L, and I332E ( “GAALIE” ) ;

vii. G236A, S239D, A330L, and I332E ( “GASDALIE” ) ;

viii. F243L, R292P, Y300L, V305I, and P396L ( “LPLIL” ) ;

ix. M252Y, S254T, and T256E ( “YTE” ) ;

x. T307A, E380A, and N434A ( “AAA” ) ;

xi. M428L and N434S ( “LS” ) ;

xii. M252Y and T256D ( “YD” ) ;

xiii. T256D and T307Q ( “DQ” ) ;

xiv. T256D and T307W ( “DW” ) ;

xv. P329G, L234A, and L235A ( “LALA-PG” ) ; and

xvi. L234A and L235A ( “LALA” ) ;

wherein numbering is according to the EU index as in Kabat.

Embodiment 49. A nucleic acid combination comprising a first nucleic acid encoding the first polypeptide and a second nucleic acid encoding the second polypeptide of any one of Embodiments 1-48.

Embodiment 50. A nucleic acid encoding both the first polypeptide and the second polypeptide of any one of Embodiments 1-48.

Embodiment 51. A vector combination comprising a first vector comprising a nucleic acid encoding the first polypeptide and a second vector comprising a second nucleic acid encoding the second polypeptide of any one of Embodiments 1-48.

Embodiment 52. A vector comprising nucleic acid encoding the first and second polypeptides of any one of Embodiments 1-48.

Embodiment 53. A host cell comprising one or more recombinant nucleic acid encoding the first polypeptide and second polypeptide of any one of Embodiments 1-48.

Embodiment 54. A method of producing a heteromultimer comprising culturing the host cell of Embodiment 53 under conditions to produce the heteromultimer.

Embodiment 55. The method of Embodiment 54, further comprising recovering the heteromultimer from the cell or cell culture.

Embodiment 56. A pharmaceutical composition comprising the composition of any one of Embodiments 1-48 and a pharmaceutically acceptable carrier.

V. Sequences

Different sequences are illustrated below. Substitutions made at a wildtype location are indicated in bold and underlined.

Table 2 summarizes the sequences.

Table 2

EXAMPLES

The following examples are offered to illustrate, but not to limit the invention. One of skill in the art will recognize that the following procedures may be modified using methods known to one of ordinary skill in the art.

Example 1. CH3 Substitutions

Nucleic acids encoding for a first and second polypeptide comprising different CH3 IgG₁ substitutions were produced to evaluate heterodimer formation. The different substitutions are shown in Table 3. HD-41 is wildtype CH3 and was used as a negative control. Multiple substitutions are separated by slash marks ( "/" ) . CH3A and CH3B, as illustrated in FIG. 1, provide an IgG₁ heavy chain polypeptides (CH3A) and a half Fc (CH3B) . CH3A and CH3B comprise the substitutions shown in Table 3.

Table 3

Example 2. Polypeptide Design to Evaluate Heterodimer Formation

Heterodimer formation was evaluated by expressing nucleic acid encoding for CH3A, CH3B, and nucleic acid encoding for light chains. With respect to a dimeric CH3 complex combining CH3A and CH3B leads to 3 possibilities: CH3A: CH3A (homodimer) ; CH3A: CH3B heterodimer; and CH3B: CH3B (homodimer) .

Formation of the different combinations can readily be distinguished based on the differences in weight. Component 1 comprises a CH3A: CH3A (homodimer) and is a Fab₂ x Fc. Component 2 comprises CH3A: CH3B heterodimer and is a Fab × Fc. Component 3 comprises CH3B: CH3B (homodimer) and is a Fc. The different components have significantly different molecular weights. The relative molecular weights of the different components are: Component 1 > Component 2 > Component 3. The different Components are illustrated in FIGs. 1A-1C.

Table 4 provides the particular amino acid sequences for the Fab heavy and light chains used in the evaluation. (Carter et al. J Immunol Methods. 2001. 248 (1–2) : 7–15) . The amino acid sequence of the Fab heavy chain polypeptide is provided by SEQ ID NO: 1 and the amino acid sequence of the Fab light chain polypeptide is provided by SEQ ID NO: 2.

Table 4

Example 3. DNA Constructs

Nucleic encoding for polypeptides described in Example 2 were designed starting with nucleic acid encoding for SP34 (Carter et al. J Immunol Methods. 2001. 248 (1–2) : 7–15) . Encoding sequence sequences were codon-optimized (Biointron) . PCR primers were designed for each fragment and synthesized by Genewiz. Target genes encoding heavy chains, light chains, a heavy chain constant region with a Kozak sequence, an N-terminal signal peptide, and a C-terminal stop codon were synthesized and subcloned into digested mammalian expression vector-pcDNA3.4 using the Gibsonmethod (Gibson, D., Young, L., Chuang, RY. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009. 6: 343–345) .

Expression vectors encoding the polypeptide sequences were transformed simultaneously into chemically competent E. coli DH5-alpha cells (NEB or similar) for plasmid preparation. After validation by Sanger sequencing (Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec. 74 (12) : 5463-7) , positive colonies were inoculated in LB (Luria Broth) medium culture with appropriate antibiotic. Desired amounts of plasmids were purified from cell pellets using a plasmid preparation kitDNA concentration was determined by UV-Vis (ultraviolet-visible) absorbance measurement at 260 nm.

Example 4. Polypeptide Expression

ExpiCHO-S^TM cells were used for transient expression with a commercially available transfection kit and culture medium (ThermoFisher Scientific) . A high-throughput transfection protocol was used on a Hamilton STAR liquid handling station. Generally, plasmids were mixed and incubated with ExpiFectamine^TM CHO reagent and OptiPRO^TM medium, and the mixture was added to the cells (viable cell density at 4-6E6 cells/mL) in a 24-deep-well plate. The final concentration of the plasmid mixture was 1 μg/mL. Transfected cells were shaken at 37℃ with 80%relative humidity and 8%CO₂. One day post-transfection, ExpiFectamine^TM enhancer and feed were supplemented and the temperature was shifted to 32℃. Cells were harvested on day 7 post-transfection.

Example 5. Purification

High-throughput purification was performed on a Hamilton STAR liquid handling station, using Tip columns containing Protein A resin (MicroSep Biological Science) . Cell culture was clarified by plate centrifugation. Clarified supernatant was loaded into a Tip column 10-20 times by repeated aspirating and dispensing. The columns were washed with neutral pH buffer (PBS (phosphate-buffered saline) ) . Polypeptides were then eluted by 5-10 times by repeated aspirating and dispensing into a clean deep-well plate with low pH buffer (50 mM NaAc, pH 3.5) . Eluted polypeptides were then titrated to neutral pH by the addition of high pH buffer (1 M Arg, pH 9.0) . Protein concentration was quantified by UV-Vis absorbance measurement at 280 nm.

Example 6. Purity Analysis

The relative amounts of Components 1, 2, and 3, produced and purified as described in Examples 1-5 were further analyzed by CE-SDS and HPLC.

SEC-UPLC (Size-Exclusion Chromatography-Ultra-High Performance Liquid Chromatography)

SEC-UPLC (Vanquish^TM Flex^TM, Thermo Fisher Scientific) was performed for aggregation measurement. Specifically, 50 μL purified antibody sample was loaded to a size exclusion column (Unix^TM-C SEC-300) , with PBS used as mobile phase and flow rate set at 0.3 mL/min Total run time for each sample was set at 10 min. The correctly paired BsAbs peaks were identified based on retention time and purity of the BsAbs was quantified as relative peak area (%) .

The CE-SDS protocol: CE-SDS (GXII Touch^TM HT, Perkin Elmer) was used to analyze by-products. The non-reduced samples were prepared by mixing 2 μL purified antibody with 7 μL SDS loading solution (HT Protein Express Sample Buffer, Perkin Elmer, with 1%SDS (sodium dodecyl sulfate) and 12.5 mM NEM (N-ethylmaleimide) ) . Non-reduced samples were heated at 70℃ for 10 min and then mixed with an additional amount of 35 μL H₂O. Prepared polypeptide samples were transferred to a 96-well plate and loaded into theProtein Express Chips were prepared for the sample runs according to theuser manual (Protein Express Assay Quick Guide, Perkin Elmer) .

Results: The results with respect to the HD-41 control are shown in FIG. 2 and Table 5. Component 1 (Fab₂ × Fc, homodimeric) is the earliest fraction eluted followed by Component 2 (Fab × Fc, heterodimeric) , finally Component 3 (Fc, homodimer) is eluted. The elution profile of those components is illustrated in FIG. 2.

Table 5

Purity of the different components can be discerned by the percentage of main peak. For example, the purity of the heterodimeric format can be determined by integrating the main and side peaks observed, and calculating the percentage of main peak's AUC (area under curve) relative to the total AUC of all the three formats’ AUC.

The relative peak area (59.170%) was quantified as the percentage of the main peak area to the sum of all peak areas, which can be used to represent the percentage of correctly paired BsAb (heterodimer) . As summarized in Table 5, the percentage of the correctly paired BsAb was 59.170%.

Table 6 summarizes the results for different constructs, analyzed using CE-SDS and SEC-UPLC. Wildtype (HD-41) was used as a negative control.

Table 6

As illustrated in Table 6 substitutions F405V/Y407A introduced into CH3A and substitution T366W introduced into CH3B (this mutant is referred to as HD-33) can significantly improve the correct pairing rate of CH3A and CH3B as compared to wildtype (without any mutations in the CH3 domain) . In addition, further replacements of Y349T into CH3A and K360Y/H435R/Y436F into CH3B (i.e., HD-18) can further promote the formation of heterodimer of CH3A and CH3B as compared to HD-33.

Table 7 provides results of another set of experiments, evaluating heterodimer formation for different constructs. Again, substitutions of residues F405V and Y407A in one CH3 domain and T366W in the other CH3 domain significantly improved the correct pairing of CH3A and CH3B. The impact of additional modifications at different positions are also illustrated in the Table 7.

Table 7

As can be seen from Table 7, substitutions F405V and Y407A in one CH3 domain and T366W in the other CH3 domain significantly improved the correct pairing; and the introduction of additional mutations (see HD-18) further increased heterodimer formation.

In another set of experiments, the impact of changing certain substitutions was examined (e.g., 360W/Y and 366W/Y) . Substitution by either tryptophan (W) or tyrosine (Y) at positions K360 and T366 resulted in a significant improvement of the correct pairing rate of CH3A and CH3B. The results are shown in Table 8.

Table 8

While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention.

Claims

A heteromultimer comprising a heterodimeric polypeptide complex, wherein

(a) the heterodimeric polypeptide complex comprises

(i) a first modified immunoglobulin CH3 domain comprising the amino acid substitutions 405V and 407A and

(ii) a second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W or 366Y; wherein numbering is according to the EU index as in Kabat; or

(b) the heterodimeric polypeptide complex comprises:

i) a first polypeptide comprising a first modified immunoglobulin CH3 domain having at least two amino acid substitutions relative to a wildtype immunoglobulin CH3 domain at positions 349, 405 or 407, wherein the substitutions are selected from the group consisting of 349T, 349A, 349S, 349V, 349G, 405V, 405A, 405T, 405S, 405G; 407A, 407T, 407S, 407G, and 407V; and

ii) a second polypeptide comprising a second modified immunoglobulin CH3 domain having at least one amino acid substitution relative to a wildtype immunoglobulin CH3 domain at positions 366 or 360, wherein the substitution is selected from the group consisting of 366Y, 366W, 366F, 366L, 360Y, 360W, 360F, and 360L, wherein numbering is according to the EU index as in Kabat.
The heteromultimer of claim 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 405 and 407; and the second modified immunoglobulin CH3 domain comprises a substitution at position 366.
The heteromultimer of claim 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 349 and 407; and the second modified immunoglobulin CH3 domain comprises a substitution at position 360.
The heteromultimer of claim 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 349, 405 and 407; and the second modified immunoglobulin CH3 domain comprises substitutions at positions 360 and 366.
The heteromultimer of any one of claims 1-4, wherein the substitutions are selected from 349T, 405V, 407A, 366W, 366Y, 360W, and 360Y.
A heteromultimer comprising a heterodimeric polypeptide complex, wherein the dimeric polypeptide complex comprises a first polypeptide comprising a first modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, and a second polypeptide comprising a second modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, wherein:

(a) the first modified immunoglobulin CH3 domain comprises the substitutions 405V, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;

(b) the first modified immunoglobulin CH3 domain comprises the substitutions 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;

(c) the first modified immunoglobulin CH3 domain comprises the substitution 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366Y;

(d) the first modified immunoglobulin CH3 domain comprises the substitution 349T, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y;

(e) the first modified immunoglobulin CH3 domain comprises the substitutions 349T and 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y;

(f) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366W;

(g) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366Y;

(h) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360W and 366Y; or

(i) the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y, 366W, 435R and 436F;

wherein numbering is according to the EU index as in Kabat.
The heteromultimer of claim 1, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitutions 405V and 407A and the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W or 366Y.
The heteromultimer of claim 7, wherein the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W.
The heteromultimer of claim 8, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 5; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 6
The heteromultimer of claim 8, wherein the second modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 360Y.
The heteromultimer of claim 10, wherein the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T.
The heteromultimer of claim 10, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 8.
The heteromultimer of claim 11, wherein the second modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitutions 435R and 436F.
The heteromultimer of claim 13, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 11.
The heteromultimer of claim 7, wherein the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T and second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitutions 366Y and 360Y.
The heteromultimer of claim 15, wherein the first modified immunoglobulin CH3 domain polypeptide comprising a sequence with a sequence identity of at least 90%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 9.
The heteromultimer of claim 7, wherein the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T and the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitutions 366Y and 360W.
The heteromultimer of claim 17, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 10.
The heteromultimer of claim 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitution 349T and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 360Y.
The heteromultimer of claim 19, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 13; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 14.
The heteromultimer of claim 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitutions 349T and 407A and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 360Y.
The heteromultimer of claim 21, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 16; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 14.
The heteromultimer of claim 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitution 405V and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 366W.
The heteromultimer of claim 23, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 17; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%to SEQ ID NO: 6.
The heteromultimer of any one of claims 1-24, wherein the first modified immunoglobulin CH3 domain and the second first modified immunoglobulin CH3 domain are derived from a human IgG₁, IgG₂, IgG₃, or IgG₄.
The heteromultimer of any one of claims 1-24, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%with any of SEQ ID NOs: 3, 19, 20 and 21 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%with SEQ ID NOs: 3, 19, 20 or 21.
The heteromultimer of any one of claims 1-26, wherein the first polypeptide further comprises a first CH2 immunoglobulin domain joined to the amino terminus of the first CH3 immunoglobulin domain and the second polypeptide further comprises a second CH2 immunoglobulin domain joined to the amino terminus of the second CH3 amino terminus, wherein the first and second CH2 immunoglobulin domains may be the same or different.
The heteromultimer of claim 27, wherein the first CH3-CH2-hinge domain is an IgG₁ or IgG₄ and the second CH3-CH2-hinge domain is an IgG₁ or IgG₄.
The heteromultimer of any one of claims 1-28 wherein the first polypeptide further comprises a first immunoglobulin hinge region joined to the amino terminus of the first CH2 domain providing a first CH3-CH2-hinge domain and the second polypeptide further comprises a second immunoglobulin hinge region joined to the amino terminus of the second CH2 amino terminus providing a second CH3-CH2-hinge domain, wherein the first and second hinge regions are joined by one or more disulfide bonds, and wherein the first CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at least 90%to SEQ ID NO: 12 and the second CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at least 90%to SEQ ID NO: 12.
The heteromultimer of claims 1 or 6, wherein either the first polypeptide comprises the sequence of SEQ ID NO: 4 and the second polypeptide comprises the sequence of SEQ ID NO: 6; the first polypeptide comprises the sequence of SEQ ID NO: 5 and the second polypeptide comprises the sequence of SEQ ID NO: 6; the first polypeptide comprises the sequence of SEQ ID NO: 13 and the second polypeptide comprises the sequence of SEQ ID NO: 14; the first polypeptide comprises the sequence of SEQ ID NO: 16 and the second polypeptide comprises the sequence of SEQ ID NO: 14; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 8; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 9; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 10; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 11; or the first polypeptide comprises the sequence of SEQ ID NO: 17 and the second polypeptide comprises the sequence of SEQ ID NO: 6.
The heteromultimer of any one of claims 1-30, wherein the first polypeptide further comprises a first binding domain and the second polypeptide further comprises a second binding domain, wherein the first and second binding domains are different from each other.
The heteromultimer of claim 1, wherein the first polypeptide further comprises a first binding domain and the second polypeptide further comprises a second binding domain, wherein the first and second binding domains are different from each other.
The heteromultimer of claim 6, wherein the first polypeptide further comprises a first binding domain and the second polypeptide further comprises a second binding domain, wherein the first and second binding domains are different from each other.
The heteromultimer of any one of claims 31-33, wherein the first and second binding domains are independently selected from a receptor or a ligand.
The heteromultimer of any one of claims 31-33, wherein the first binding domain is a first antigen binding domain and the second binding domain is a second antigen binding domain, wherein the first and second antigen binding domains bind to different epitopes.
The heteromultimer of claim 35, wherein the first and second antigen binding domains bind to different antigens.
The heteromultimer of any one of claims 35 or 36, wherein at least one of the first and second polypeptides further comprises a third antigen binding domain.
The heteromultimer of claim 37, wherein the first polypeptide further comprises a third antigen binding domain, and the second polypeptide further comprises a fourth antigen binding domain.
The heteromultimer of claim 38, wherein the first polypeptide further comprises a fourth antigen binding domain.
The heteromultimer of any one of claims 33-39, wherein each antigen binding domain is selected from the group consisting of a VH, VL, VHH (sdAb or dAb) , Fab, Fab’, F (ab’) ₂, scFab, scFv, (scFv) ₂, Fv, taFv, DART and bispecific T-cell engager.
The heteromultimer of claims 33 or 34, wherein the heteromultimer is selected from the group comprising a DART-Fc, CrossMAb, DuetMab, TriFabs, and controlled Fab arm exchange generated bispecific antibodies.
The heteromultimer of any one of claims 33-41, wherein the heteromultimer binds to one, two, three, four or more antigenic polypeptides.
The heteromultimer of claim 42, wherein the antigenic polypeptides are selected from the group consisting of CD19, CD20, EpCAM, CEA, PSMA, GD2, CD30, CD38, CD47, CD52, c-Met, CA19-9, CA72-4, CAM 17.1, CD1a, CD5, CD11A, CD40, CD44, B7-H3, B7-H4, B7-H6, PD-L1 or antigenic fragments thereof.
The heteromultimer of any one of claims 1-29 or 31-43, wherein the first and/or second polypeptide further comprises one or more Fc mutations selected from the group comprising:

i. S239D and I332E ( “DE” ) ;

ii. S239D, I332E, and A330L ( “DLE” ) ;

iii. S239D;

iv. I332E;

v. S298A, E333A, and K334A;

vi. G236A, A330L, and I332E ( “GAALIE” ) ;

vii. G236A, S239D, A330L, and I332E ( “GASDALIE” ) ;

viii. F243L, R292P, Y300L, V305I, and P396L ( “LPLIL” ) ;

ix. M252Y, S254T, and T256E ( “YTE” ) ;

x. T307A, E380A, and N434A ( “AAA” ) ;

xi. M428L and N434S ( “LS” ) ;

xii. M252Y and T256D ( “YD” ) ;

xiii. T256D and T307Q ( “DQ” ) ;

xiv. T256D and T307W ( “DW” ) ;

xv. P329G, L234A, and L235A ( “LALA-PG” ) ; and

xvi. L234A and L235A ( “LALA” ) ;

wherein numbering is according to the EU index as in Kabat.
A nucleic acid combination comprising a first nucleic acid encoding the first polypeptide and a second nucleic acid encoding the second polypeptide of any one of claims 1-44.
A nucleic acid encoding both the first polypeptide and the second polypeptide of any one of claims 1-44.
A vector combination comprising a first vector comprising a nucleic acid encoding the first polypeptide and a second vector comprising a second nucleic acid encoding the second polypeptide of any one of claims 1-44.
A vector comprising nucleic acid encoding the first and second polypeptides of any one of claims 1-44.
A host cell comprising one or more recombinant nucleic acid encoding the first polypeptide and second polypeptide of any one of claims 1-44.
A method of producing a heteromultimer comprising culturing the host cell of claim 49 under conditions to produce the heteromultimer.
The method of claim 50, further comprising recovering the heteromultimer from the cell or cell culture.
A pharmaceutical composition comprising the composition of any one of claims 1-45 and a pharmaceutically acceptable carrier.