WO2020205504A1

WO2020205504A1 - Common light chains and methods of use

Info

Publication number: WO2020205504A1
Application number: PCT/US2020/025189
Authority: WO
Inventors: Jason Michael LAJOIE; Michael March SCHMIDT
Original assignee: Compass Therapeutics Llc
Priority date: 2019-03-29
Filing date: 2020-03-27
Publication date: 2020-10-08

Abstract

The present disclosure provides methods of generating multispecific antigen-binding molecules comprising a common light chain compatible with two or more different heavy chains. The present disclosure further provides methods of identifying common light chains that are compatible with two or more different heavy chains

Description

COMMON LIGHT CHAINS AND METHODS OF USE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

62/826,535 filed March 29, 2019 entitled Common Light Chains and Methods of Use the contents of which are herein incorporated by reference in its entirety.

REFERENCE TO THE SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 2183_1046PCT_SEQLST.txt, was created on March 27, 2020, and is 9,118 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0003] The disclosure relates to methods of generating multispecific antigen-binding molecules comprising a common light chain compatible with two or more different heavy chains. BACKGROUND OF THE DISCLOSURE

[0004] Multispecific antigen-binding molecules (ABMs) combine two or more different antigen binding units (ABUs) in a single molecule, facilitating a multifaceted targeting approach, novel mechanisms of action, and potentially higher clinical efficacies. While multispecific ABMs have several advantages as compared to a monospecific ABM, many multispecific ABMs are associated with issues of production, stability and pharmacokinetic properties. One means to address these issues is to pair a common light chain with each of the different heavy chains in the ABUs of the ABM. However, utilization of some common light chains may negatively impact the binding affinity of one or more of the ABUs for one or more target epitopes. In some cases, researchers have attempted to address the reduced binding of an ABU associated with a specific common light chain by mutating and optimizing the variable heavy domain of the ABU. See, e.g., Jackman, et al, 2010, American Society for Biochemistry and Molecular Biology, 20850- 20859; van Blarcom et al., 2018, MABs, 10:256-268; Krah et al, 2017, Protein Eng Des Sel, 30:290-301; Geuijen et al., 2018, Cancer Cell, 922-936. Alternatively, some researchers have attempted to resolve the issues associated with common light chains in multispecific ABMs by identifying a candidate common light chain from multiple shuffle libraries, and then performing several iterative rounds of mutagenesis on the candidate light chain and the different heavy chains of the ABM in order to optimize the binding affinity and other properties of the multispecific ABM. See, e.g. , Sampei et al, 2013, PLoS One, p.e57479; and Shiraiwa et al., 2019, Methods, 10-20. However, these existing methods of optimizing a common light chain are extremely laborious and time-consuming in that many rounds of mutagenesis and optimization are needed before the target affinity is achieved or before an appropriate common light chain is obtained. These methods also design new common light chains for a multispecific ABM on a case- by-case basis, thereby requiring new rounds of experimentation/optinuzation to be performed for each new ABU heavy chain/common light chain pairing. As such, there remains a need for more effective methods of identifying common light chains that are compatible with one or more heavy chains.

SUMMARY OF THE DISCLOSURE

[0005] In some embodiments, the disclosure provides for a method for generating a multispecific antigen-binding molecule (ABM) comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain amino acid sequences; wherein the variant or mutated light chain amino acid sequence is identified as being compatible if: i) when paired with a first heavy chain amino acid sequence to generate a first antigen-binding unit (ABU), the first ABU binds a first epitope and is associated with a first desired characteristic; and li) when paired with a second heavy' chain amino acid sequence to generate a second ABU, the second ABU binds a second epitope and is associated with a second desired characteristic; and b) generating a multispecific ABM comprising the first ABU and the second ABU In some embodiments, the disclosure provides for a method for generating a multispecific antigen-binding molecule (ABM) comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain ammo acid sequences; wherein a variant or mutated light chain amino acid sequence is identified as being compatible if: i) when paired with a first heavy chain amino acid sequence to generate a first antigen-bmding unit (ABU), the first ABU is associated with a desired binding affinity to a first epitope; and li) when paired with a second heavy chain amino acid sequence to generate a second ABU, the second ABU is associated with a desired binding affinity to a second epitope; and b) generating a multispecific ABM comprising the first ABU and the second ABU. In some embodiments, the disclosure provides for a method for generating a multispecific antigen- binding molecule (ABM) comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain ammo acid sequences; wherein a variant or mutated light chain am o acid sequence is identified as being compatible if: i) when paired with a first heavy chain ammo acid sequence to generate a first antigen-binding unit (ABU), the first ABU binds a first epitope and is associated with a first desired characteristic; and u) when paired with a second heavy chain amino acid sequence to generate a second ABU, the second ABU binds a second epitope and is associated with a second desired characteristic; and b) generating a multispecific ABM comprising the first ABU and the second ABU; wherein the complementarity determining regions (CDRs) of the multispecific ABM are not further optimized for the desired characteristic. In some embodiments, the disclosure provides for a method for generating a multispecific antigen-binding molecule (ABM) comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain amino acid sequences; wherein the variant or mutated light chain amino acid sequences in the Database were mutated as compared to a reference light chain ammo acid sequence; wherein a variant or mutated light chain ammo acid sequence is identified as being compatible if: i) when paired with a first heavy chain amino acid sequence to generate a first antigen-binding unit (ABU), the first ABU binds a first epitope and is associated with a first desired characteristic; and ii) when paired with a second heavy chain amino acid sequence to generate a second ABU, the second ABU binds a second epitope and is associated with a second desired characteristic; and b) generating a multispecific ABM comprising the first ABU and the second ABU. In some embodiments, the variant or mutated light chain ammo acid sequences in the Database were mutated as compared to a reference light chain ammo acid sequence. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 1. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 4. In some embodiments, the Database comprises a plurality of variant or mutated light chain ammo acid sequences, wherein each of the variant or mutated light chain ammo acid sequences in the plurality of sequences comprises at least one ammo acid alteration as compared to a CDR1, CDR2 and/or CDR3 of a reference light chain ammo acid sequence. In some embodiments, the Database comprises a plurality of variant or mutated light chain ammo acid sequences, wherein each of the variant or mutated light chain am o acid sequences in the plurality of sequences comprises at least one ammo acid alteration as compared to the ammo acid sequences of any one of SEQ ID NQs: 5-19. In some embodiments, the Database comprises a plurality of variant or mutated light chain ammo acid sequences, wherein each of the variant or mutated light chain amino acid sequences in the plurality of sequences comprises at least one amino acid alteration in the framework region as compared to the framework region of a reference light chain amino acid sequence. In some embodiments, the variant or mutated light chain ammo acid sequences in the Database were mutated as compared to a single reference light chain amino acid sequence. In some

embodiments, one or more of the variant or mutated light chain amino acid sequences in the Database were determined using next generation sequencing. In some embodiments, the first and/or second desired characteristic is desired binding affinity. In some embodiments, the variant or mutated light chain ammo acid sequences were generated using single-site saturation. In some embodiments, the variant or mutated light chain amino acid sequences were

characterized using deep mutational scanning. In some embodiments, the Database comprises variant or mutated light chain amino acid sequences consisting of 1-5 mutated amino acid residues as compared to a reference light chain ammo acid sequence. In some embodiments, the Database comprises variant or mutated light chain amino acid sequences consisting of one mutated amino acid residue as compared to a reference light chain amino acid sequence. In some embodiments, each of the variant or mutated light chain amino acid sequences in the Database differs from the other variant or mutated light chain ammo acid sequences in the Database by no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acids. In some embodiments, the Database comprises at least 50 different variant or mutated light chains amino acid sequences. In some embodiments, the Database comprises at least 100 different variant or mutated light chains ammo acid sequences. In some embodiments, the Database comprises at least 200 different variant or mutated light chains ammo acid sequences. In some embodiments, the Database comprises at least 500 different variant or mutated light chains amino acid sequences. In some embodiments, the first desired characteristic is a desired binding affinity of the first ABU for a first epitope. In some embodiments, the second desired characteristic is a desired binding affinity of the second ABU for a second epitope. In some embodiments, the desired binding affinity of the first ABU for the first epitope is measured in terms of an enrichment ratio (ER). In some embodiments, the desired binding affinity of the second ABU for the second epitope is measured in terms of an enrichment ratio (ER). In some embodiments, the desired binding affinity of the first ABU for the first epitope is measured in terms of an enrichment ratio (ER), and wherein the desired binding affinity of the second ABU for the second epitope is measured in terms of an enrichment ratio. In some embodiments, the variant or mutated light chain is identified as being a compatible light chain if the first ABU has an ER greater than I, 1.5, 2, 2.5, 3, 3.5 or 4 for the first epitope, and the second ABU has an ER greater than -1 5, -1.0, -0.5, 0, 0.5 or 1 for the second epitope. In some embodiments, the variant or mutated light chain is identified as being a compatible light chain if the first ABU has an ER greater than 2 for the first epitope, and the second ABU has an ER greater than -1.5 for the second epitope. In some embodiments, the variant or mutated light chain is identified as being a compatible light chain if the first ABU has an ER greater than 2 for the first epitope, and the second ABU has an ER greater than -2 for the second epitope. In some embodiments, the first heavy chain was affinity matured for binding to the first epitope. In some embodiments, the second heavy chain was affinity matured for binding to the second epitope. In some

embodiments, the disclosure provides for a method for generating a multispecific antigen binding molecule (ABM) comprising a first antigen-binding unit (ABU) having a first desired characteristic and a second ABU having a second desired characteristic, the method comprising: a) screening a library of first ABUs for a first desired characteristic, wherein the library comprises a plurality of first ABUs, wherein each of the first ABUs comprises: i) the same first heavy chain amino acid sequence and ii) a unique variant or mutated light chain amino acid sequence relative to a reference light chain ammo acid sequence; and b) generating a multispecific ABM comprising: 1) a first ABU identified from step (a) having the desired characteristic; and 2) a second ABU, wherein the second ABU has a second desired

characteristic and comprises: (x) a second heavy chain ammo acid sequence and (y) the same variant light chain amino acid sequence as the first ABU. In some embodiments, each variant or mutated light chain amino acid sequence comprises one or more amino acid substitutions, deletions, or insertions relative to a reference light chain am o acid sequence. In some embodiments, each variant or mutated light chain ammo acid sequence comprises fewer than 20, fewer than 15, fewer than 10, fev/er than 5; no more than five, 10, 15, or 20 amino acid substitutions, deletions, or insertions relative to a reference light chain amino acid sequence. In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 3. In some embodiments, the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 4. In some embodiments, a plurality of the variant or mutated light chain ammo acid sequences in the library comprise at least one amino acid alteration as compared to a CDR1 , CDR2 and/or CDR3 of a reference light chain ammo acid sequence. In some embodiments, a plurality of the variant or mutated light chain ammo acid sequences in the library comprise at least one amino acid alteration as compared to the framework region of a reference light chain ammo acid sequence. In some embodiments, a plurality of the variant or mutated light chain amino acid sequences in the library comprises at least one ammo acid alteration as compared to the ammo acid sequences of any one of SEQ ID NQs: 5-19. In some embodiments, the variant or mutated light chain amino acid sequences in the library were mutated as compared to a single reference light chain amino acid sequence. In some embodiments, the variant or mutated light chain ammo acid sequences were generated using single-site saturation. In some embodiments, the variant or mutated light chain amino acid sequences were characterized using deep mutational scanning. In some embodiments, the screening step a) comprises phage display, yeast display, and/or mammalian display. In some embodiments, the screening step of a) comprises assaying the binding affinity of the first ABUs for a first antigen. In some embodiments, the binding affinity of the first ABUs is compared to the binding affinity of a reference antibody or antigen-bmding fragment for the same antigen. In some embodiments, the screening step a) comprises panning and/or sorting the first ABUs based on whether the first ABUs are determined to have the desired characteristic. In some embodiments, the method further comprises determining the sequence of at least a portion of a first ABU determined to have the first desired characteristic, or determining the nucleotide sequence of at least a portion of the nucleic acid encoding the first ABU determined to have the first desired characteristic. In some

embodiments, next generation sequencing is used to determine the nucleotide sequence of at least a portion of the nucleic acid encoding the first ABU. In some embodiments, the first desired characteristic is a desired binding affinity for a first epitope, and wherein the second desired characteristic is a desired binding affinity for a second epitope. In some embodiments, the desired binding affinity of the first ABU for the first epitope is measured in terms of an enrichment ratio (ER), and wherein the desired binding affinity of the second ABU for the second epitope is measured in terms of an enrichment ratio. In some embodiments, the first ABU is determined to have the desired characteristic if the first ABU has an ER greater than 1 , 1.5, 2, 2 5, 3, 3.5 or 4 for the first epitope, and the second ABU is determined to have the desired characteristic if the second ABU has an ER greater than -1.5, -1.0, -0.5, 0, 0.5 or 1 for the second epitope. In some embodiments, the first ABU is determined to have the desired characteristic if the first ABU has an ER greater than 2 for the first epitope, and the second ABU is determined to have the desired characteristic if the second ABU has an ER greater than -1 .5 for the second epitope. In some embodiments, the first ABU is determined to have the desired characteristic if the first ABU has an ER greater than 2 for the first epitope, and the second ABU is determined to have the desired characteristic if the second ABU has an ER greater than -2 for the second epitope. In some embodiments, the first heavy chain was affinity matured for binding to the first epitope. In some embodiments, the second heavy chain was affinity matured for binding to the second epitope. In some embodiments, data obtained from the screening step a) are stored m a Database. In some embodiments, the method further comprises generating an expression vector comprising one or more nucleotide sequences encoding the multispecific ABM. In some embodiments, the method further comprises expressing the ABM from a cell.

[0006] In some embodiments, the disclosure provides for a multispecific ABM generated using any of the methods disclosed herein. In some embodiments, the multispecific ABM is a bispecific ABM. In some embodiments, the multispecific ABM is a trispecific ABM. In some embodiments, the ABM is human or humanized. In some embodiments, the ABM is an antibody.

[0007] In some embodiments, the disclosure provides for a nucleic acid or plurality of nucleic acids encoding any of the ABM disclosed herein.

[0008] In some embodiments, the disclosure provides for a vector comprising the nucleic acid or plurality of any of the nucleic acids disclosed herein.

[0009] In some embodiments, the disclosure provides for a cell comprising any of the nucleic acids or plurality of nucleic acids disclosed herein or any of the vectors disclosed herein.

[0010] In some embodiments, the disclosure provides for a method for creating a Database of variant or mutated light chains, comprising: a) providing a library comprising a plurality of different variant or mutated light chains; wherein each variant or mutated light chain in the library' comprises at least one ammo acid alteration as compared to a reference light chain amino acid sequence; b) providing a set of heavy chains, wherein each of the heavy chains comprises the same amino acid sequence; c) separately pairing each of the different variant or mutated light chains with heavy chains from the set of heavy chains, thereby generating a library of antigen binding units (ABUs) that bind an antigen; d) screening the ABUs from the library of ABUs for a desired characteristic, e) optionally repeating steps c) and d) with a different set of heavy chains each having an identical amino acid sequence; f) storing in a Database the data obtained from the screening step d) and optionally from the screening step in e). In some embodiments, each variant light chain amino acid sequence comprises one or more ammo acid substitutions, deletions, or insertions relative to a reference light chain amino acid sequence. In some embodiments, each variant light chain amino acid sequence comprises fewer than 20, fev/er than 15, fewer than 10, fewer than 5; no more than five, 10, 15, or 20 amino acid substitutions, deletions, or insertions relative to a reference light chain ammo acid sequence. In some embodiments, the reference light chain annuo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 3. In some embodiments, the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a plurality of the variant or mutated light chain amino acid sequences in the library comprise at least one amino acid alteration as compared to a CDRl, CDR2 and/or CDR3 of a reference light chain ammo acid sequence. In some embodiments, a plurality of the variant or mutated light chain amino acid sequences in the library comprise at least one amino acid alteration as compared to the framework regions of a reference light chain ammo acid sequence. In some embodiments, a plurality of the variant or mutated light chain amino acid sequences in the library comprises at least one amino acid alteration as compared to the amino acid sequences of any one of SEQ ID NOs: 5-19. In some embodiments, the variant or mutated light chain ammo acid sequences in the library were mutated as compared to a single reference light chain amino acid sequence. In some embodiments, the variant or mutated light chain amino acid sequences were generated using single-site saturation. In some embodiments, the variant or mutated light chain amino acid sequences were characterized using deep mutational scanning. In some embodiments, the screening step d) and/or e) comprises phage display, yeast display, and/or mammalian display. In some embodiments, the screening step of d) comprises assaying the binding affinity of the ABUs for a first antigen. In some embodiments, the binding affinity of the ABUs in step d) are compared to the binding affinity of a reference ABU for the same antigen. In some embodiments, the screening step d) comprises panning and/or sorting the ABUs based on whether the ABUs are determined to have the desired characteristic. In some embodiments, the method further comprising step e), wherein step e) comprises repeating steps c) and d) with a different set of heavy chains each having an identical amino acid sequence. In some embodiments, the heavy chains of step b) were affinity matured for binding to an epitope. in some embodiments, the heavy chains of step e) were affinity matured for binding to an epitope.

[0011] In some embodiments, the disclosure provides for a database comprising: a) a plurality of variant or mutated light chain amino acid sequences; h) a plurality of heavy chain ammo acid sequences; c) binding affinity data of a plurality of antigen-bmdmg unit (ABUs) for one or more antigens, wherein each of the ABUs comprises a variant or mutated light chain amino acid sequence selected from a) and a heavy chain amino acid sequence selected from b).

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present application includes the following figures. The figures are intended to illustrate certain embodiments and/or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0013] Figure 1 shows a simplified schematic outline of a platform for developing multispecific antigen-binding molecules.

[0014] Figure 2A provides a“heat map” of enrichment ratios (ERs) for each light chain mutation generated from parallel, common light chain, deep mutational scanning, library panning campaigns. Amino acid substitutions with positive enrichment have values greater than zero, and those with negative enrichment or depletion have values less than zero. Figure 2B shows the same ER data from Figure 2A plotted to illustrate the correlation between the Target A ER values and Target B ER values. Select compatible mutations are labeled and shown with grey- filled, thick outlined symbols and non-compatible mutations are shown as black-filled symbols. Figure 2C tabulates affinity measurements for different combinations of affinity matured variable heavy chains (“ AffMat VH”) and specific mutated light chain constructs.

[0015] Figure 3A provides a“heat map” of enrichment ratios for each light chain mutation generated from a common light chain deep mutational scanning library panning campaign against target C. Ammo acid substitutions with positive enrichment have values greater than zero, and those with negative enrichment have values less than zero. Figure 3B provides flow cytometry plots showing the sorting scheme used during an anti-D mammalian display campaign. Sorted gates are shown in each plot. Figure 3C plots the correlation of mutation ERs between Target C panning and either High or Medium affinity sorts from Target D sorting. Mutations falling within the dashed boxes are those identified by the ER cutoff analysis.

DETAILED DESCRIPTION

[0016] The following description recites various aspects and embodiments of the disclosed compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill m the art: therefore, information well known to the skilled artisan is not necessarily included.

I. DEFINITIONS

[0017] As used herein, the singular forms“a”,“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms“including”,“includes”,“having”,“has”,“with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term“comprising”.

[0018] The terms“determining”,“measuring”,“evaluating”,“assessing”,“assaying”, “analyzing”, and their grammatical equivalents can be used interchangeably herein to refer to any form of measurement and include determining if an element is present or not (for example, detection). These terms can include both quantitative and/or qualitative determinations.

Assessing may be relative or absolute.

[0019] In general,“sequence identity” refers to an exact nucleotide-to-nucleotide or amino aeid-to-ammo acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by- determining their“percent identity.” The percent identity to a reference sequence (e.g., an amino acid sequence) may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Conservative substitutions are not considered as matches when determining the number of matches for sequence identity. It wall be appreciated that where the length of a first sequence (A) is not equal to the length of a second sequence (B), the percent identity of A:B sequence wall be different than the percent identity of B:A sequence. Sequence alignments, such as for the purpose of assessing percent identity, may be performed by any suitable alignment algorithm or program, including, but not Imuted to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available on the world wide web at ebi.ac.uk/Tools/psa/emboss_needle/), the BLAST algorithm (see, e.g., the BLAST alignment tool available on the world wade web at

blast.ncbi.nlm.nih.gov/Blast.cgi), the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available on the world wide web at ebi.ac.uk/Tools/psa/emboss_water/), and Clustal Omega alignment program (see e.g., the world wide web at clustal.org/omega/ and F. Sievers et ah, Mol Sys Biol. 7: 539 (2011)). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters. The BLAST program is based on the alignment method of Karim and Altschui, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschui, et al., J. Mol. Biol. 215:403-410 (1990); Karim and

Altschui, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschui et al., Nucleic Acids Res. 25:3389-3402 (1997).

[0020] As used herein, the term“desired characteristic” refers to a particular light chain, heavy chain, and/or ABU characteristic for which a screen can be designed to recognize the presence or absence of that characteristic in a plurality of light chains, heavy chains and/or ABLJs. In particular embodiments, the desired characteristic is a binding affinity of an ABU for a specific epitope. In some embodiments, the desired characteristic is an ER of an ABU following a sorting process (e.g., a binding step). In some embodiments, the sorting process is a binding affinity assay. In some embodiments, the desired characteristic of an ABU is attributable to the light chain component of the ABU.

[0021] As used herein,“not optimized further” or“not further optimized” means no amino acid alterations (e.g., substitution, insertion or deletion) are made to a specific amino acid sequence. In some embodiments, any of the light chains, heavy chains, and/or ABUs obtained using any of the methods disclosed herein is not optimized further for the desired characteristic. However, the light chain, heavy chain, or ABU can be further optimized to improve, for example, manufacturing and/or purification. For the avoidance of doubt,“not optimized further” does not include post-translational modifications (e.g., PEGylation, HESYLATION^®, acetylation, glycosylation). In some embodiments, the complementarity-determining regions (CDRs) of an ABU generated using any of the methods disclosed herein is not optimized further following the pairing of a heavy chain with a compatible light chain that was identified from a Database of variant or mutated light chains. In some embodiments, the framework regions of the variable domains of an ABU generated using any of the methods disclosed herein is not optimized further following the pairing of a heavy chain with a compatible light chain that was identified from a Database of variant or mutated light chains. In some embodiments, an ABU generated according to any of the methods disclosed herein comprises one or more constant domains, wherein the constant domain(s) of the ABU can be further altered/optimized. In some embodiments, the constant domam(s) of the ABU can be further altered m the process of isotype s witching. In some embodiments, the constant domain! s) of the ABU can be further altered to facilitate“knob into holes” amino acid alterations. As used herein, the term“step,” when used in reference to a method, is intended to include the plural form“steps” as well, unless the context clearly indicates otherwise. For example, a screening step in a method may comprise multiple stages to complete.

[0022] As used herein, the term“binds” or“binding to” when used in reference to an

ABM-' ABU that“binds” an epitope/antigen means that the ABMABU is capable of binding or has the ability to bind to the epitope/antigen under physiological conditions, and does not require that the ABM/ ABU binds to the epitope/antigen at any particular period in time. For example, in some embodiments, the disclosure provides for a composition comprising a multispecific ABM that binds to a first epitope and a second epitope, but the composition is not required to include any of the first epitope or second epitope in the composition. However, if, for example, the first epitope was added to th e composition, the multi specific ABM would be capable of binding to the first epitope under physiological conditions.

[0023] As used herein, the term“associated with” when used in reference to a characteristic (e.g., binding affinity, enrichment ratio) of a light chain, heavy chain, ABU, and/or ABM, means that the light chain, heavy chain, ABU, and/or ABM has the specific characteristic under the appropriate conditions ( e.g physiological conditions). For example, an ABU or ABM may be associated with a specific binding affinity for a target epitope in the presence or absence of the target epitope, and the binding affinity associated with the ABU and ABM may be

observed/measured when the ABU or ABM is mixed with the target epitope under physiological conditions. 11. COMPOSITIONS

Light Chains

j0024J In some embodiments, the disclosure provides for an ABM or ABU comprising any of the light chains disclosed herein. As used herein, the term“light chain” includes, but is not limited to, variant or mutated light chains, common light chains, and reference light chains. In some embodiments, the light chain is a common light chain. In some embodiments, the light chain is a variant or mutated light chain. In some embodiments, the variant or mutated light chain has been mutated in comparison to a reference light chain amino acid sequence. Reference light chain amino acid sequences include, for example, human germline light chain amino acid sequences. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-4. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3.

In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 4. In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of or combination of SEQ ID NOs: 5-7. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of or combination of SEQ ID NOs: 8-10. In some embodiments, the reference light chain ammo acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of or combination of SEQ ID NOs: 11-13. In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of or combination of SEQ ID NOs: 14-16. In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of or combination of SEQ ID NOs: 17-19. In some embodiments, the variant or mutated light chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ammo acid alterations (e.g., substitutions, insertions, and/or deletions) as compared to a reference light chain ammo acid sequence. In some embodiments, the variant or mutated light chain comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations (e.g., substitutions, insertions, and/or deletions) as compared to a reference light chain amino acid sequence. In some embodiments, the variant or mutated light chain comprises less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 5, less than 4, less than 3, less than 2, or only 1 amino acid alteration (e.g., substitution, insertion, and/or deletion) as compared to a reference light chain amino acid sequence. In some embodiments, the amino acid alteration(s) is in one or more complementarity-determining regions (CDRs) of the reference light chain amino acid sequence. In some embodiments, the amino acid alteration(s) is in CDR1 of the reference light chain amino acid sequence. In some embodiments, the amino acid alteration(s) is in CDR2 of the reference light chain amino acid sequence. In some

embodiments, the amino acid alteration(s) is in CDR3 of the reference light chain amino acid sequence. In some embodiments, the amino acid alteration(s) is in the framework region of the reference light chain ammo acid sequence. In some embodiments, the amino acid alteration(s) is one or more an amino acid substitutions. In some embodiments, the amino acid aiteration(s) is one or more deletions. In some embodiments, the amino acid alteration(s) is one or more insertions. In some embodiments, the variant or mutated light chain amino acid sequence comprises no more than 1 amino acid alteration (e.g., amino acid substitutions, insertions or deletions) as compared to a single reference light chain amino acid sequence. In some embodiments, the no more than 1 amino acid alteration (e.g., ammo acid substitution, insertion or deletion) occurs in the variable region of the single reference light chain amino acid sequence. In some embodiments, the no more than 1 ammo acid alteration (e.g., am o acid substitution, insertion or deletion) occurs in a CDR of the single reference light chain ammo acid sequence. | 0025] In some embodiments, the variant or mutated light chain was characterized by utilizing deep mutational scanning of a reference light chain ammo acid sequence. In some embodiments, the variant or mutated light chain was generated by utilizing single site saturation mutagenesis.

[0026] In some embodiments, the light chain is a common light chain that has been determined to be compatible with two or more different heavy chains. In some embodiments, the common light chain is any of the variant or mutated light chains disclosed herein.

[0027] As used herein, the terms“common light chain” or“compatible light chain” mean a light chain that is compatible with two or more different heavy chains. In some embodiments, any of the variant or mutated lights disclosed herein are identified as being a common light chain or a compatible light chain. In some embodiments, the variant or mutated light chain is “compatible with” a first (or second or further) heavy chain if an ABU generated from the variant or mutated light chain and the first (or second or further) heavy chain is associated with a desired characteristic. In some embodiments, the variant or mutated light chain is“compatible with” a first (or second or further) heav ' chain if an ABU generated from the variant or mutated light chain and the first (or second or further) heavy chain is associated with a binding affinity for a target epitope that is at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 2 -fold, at least 2.5 fold, at least 3 -fold, at least 3.5 fold, at least 4-fold, at least 4.5 fold, least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250- fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, least 1000-fold, at least 5000-fold, at least 10, 000-fold, or more, greater than the binding affinity for the same target epitope of an ABU generated from the reference light chain ammo acid sequence and the first (or second or further) heavy chain. In some embodiments, the variant or mutated light chain is“compatible with” a first (or second or further) heavy chain if an ABU generated from the variant or mutated light chain, and the first (or second or further) heavy chain is associated with a binding affinity for a target epitope that is not less than 200%, not less than 150%, not less than 100%, not less than 75%, not less than 50%, not less than 25%, or not less than 10% of the binding affinity for the same target epitope of an ABU generated from the reference light chain amino acid sequence and the first (or second or further) heavy chain. In particular embodiments, the variant or mutated light chain is “compatible with” a first (or second or further) heavy chain if an ABU generated from the variant or mutated light chain and the first (or second or further) heavy chain is associated with a binding affinity for a target epitope that is not less not less than 50%, not less than 25%, or not less than 10% of the binding affinity for the same target epitope of an ABU generated from the reference light chain ammo acid sequence and the first (or second or further) heavy chain.

]0028J In some embodiments, a variant or mutated light chain is“compatible with” a first (or second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and a first (or second or further) heavy chain have a higher frequency following a sorting process than a plurality⁷ of ABUs comprising a reference light chain amino acid sequence and the first (or second or further) heavy chain. In some embodiments, a variant or mutated light chain is“compatible with” a first (or second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and the first (or second or further) heavy chain have at least a 10%, 20%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900% or 1000% higher frequency in a population of ABUs following a sorting process than a plurality of ABUs comprising a different variant or mutated light chain amino acid sequence and the first (or second or further) heavy chain in the same population of ABUs following the same sorting process. In some embodiments, the variant or mutated light chain is“compatible with” a first (or second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and a first (or second or further) heavy chain has a frequency in a population of ABUs following a sorting step that is not less than 200%, not less than 150%, not less than 100%, not less than 75%, not less than 50%, not less than 25%, or not less than 10% of the frequency of an ABU comprising a reference light chain ammo acid sequence and the first (or second or further) heavy chain in a population of ABUs following the same sorting step. In some embodiments, the sorting step comprises a binding assay step. In some embodiments, the sorting step comprises phage panning (e.g., using Ml 3 helper-phage) with a particular epitope of interest. In some embodiments, the sorting step comprises mammalian display sorting (e.g., display on HEK cells) with a particular epitope of interest.

[0029] In some embodiments, the variant or mutated light chain is“compatible with” a first (or second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and a first (or second or further) heavy chain has at least a 10%, 20%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900% or 1000% greater enrichment ratio following a sorting step than an ABU comprising a reference light chain amino acid sequence and the first (or second or further) heavy chain following the same sorting step in some embodiments, the variant or mutated light chain is“compatible with” a first (or second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and a first (or second or further) heavy chain has an enrichment ratio following a sorting step that is not less than 200%, not less than 150%, not less than 100%, not less than 75%, not less than 50%, not less than 25%, or not less than 10% of the enrichment ratio of an ABU comprising a reference light chain amino acid sequence and the first (or second or further) heavy chain following the same sorting step. In some embodiments, a variant or mutated light chain is identified as being a compatible light chain if a first ABU comprising a first heavy chain and the variant or mutated light chain has an ER greater than 1, 1.5, 2, 2.5, 3, 3.5 or 4 for a first epitope, and a second ABU comprising a second heavy chain and the variant or mutated light chain has an ER greater than -1.5, -1.0, -0.5, 0, 0.5 or 1 for a second epitope. In some embodiments, a variant or mutated light chain is identified as being a compatible light chain if a first ABU comprising a first heavy chain and the variant or mutated light chain has an ER greater than or equal to 2 for a first epitope, and a second ABU comprising a second heavy chain and the variant or mutated light chain has an ER greater than or equal to -1.5 for a second epitope. In some embodiments, a variant or mutated light chain is not identified as being a compatible light chain if a first ABU comprising a first heavy chain and the variant or mutated light chain has an ER greater than 2, but a second ABU comprising a second heavy chain and the variant or mutated light chain has an ER less than -2, In some embodiments, the enrichment ratio (ER) is calculated according to the following formula: ER=log2(Fi/Fo) where Fi is the ABU frequency after i rounds of sorting and Fo is the frequency of the same ABU in the starting plurality of ABUs.

[0030] In some embodiments, the variant or mutated light chain is compatible with (as defined herein) a first heavy chain, and is also compatible with (as defined herein) a second heavy chain. In some embodiments, the variant or mutated light chain is compatible with (as defined herein) a first heavy chain, a second heavy chain, and a third heavy chain. In some embodiments, the variant or mutated light chain is compatible wath (as defined herein) a first heavy chain, a second heavy chain, a third heavy chain, and a fourth heavy chain. In some embodiments, the variant or mutated light chain is compatible with the first heavy chain if an ABU generated from the variant or mutated light chain and the first heavy chain is associated with a binding affinity for a target epitope that is at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 2-fold, at least 2.5 fold, at least 3-fold, at least 3.5 fold, at least 4- fold, at least 4.5 fold, least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60- fold, at least 70-fold, least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600- fold, at least 700-fold, at least 800-fold, at least 900-fold, least 1000-fold, at least 5000-fold, at least 10,000-fold, or more, greater than the binding affinity for the same target epitope of an ABU generated from the reference light chain amino acid sequence and the first heavy chain: and the variant or mutated light chain is compatible with the second (or further) heavy chain if an ABU generated from the variant or mutated light chain and the second heavy chain is associated with a binding affinity_' for a target epitope that is at least 10%, 20%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900% or 1000% greater than the binding affinity for the same target epitope of an ABU generated from the reference light chain amino acid sequence and the second heavy chain. In some embodiments, the variant or mutated light chain is compatible with the first heavy chain if an ABU generated from the variant or mutated light chain and the first heavy chain is associated with a binding affinity' for a target epitope that is at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 2- fold, at least 2.5 fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 4.5 fold, least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20- fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, least 80- fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, least 1000-fold, at least 5000-fold, at least 10,000-fold, or more, greater than the binding affinity for the same target epitope of an ABU generated from the reference light chain amino acid sequence and the first heavy chain; and the variant or mutated light chain is compatible with the second (or further) heavy chain if an ABU generated from the variant or mutated light chain and the second heavy chain ABU is associated with a binding affinity for a target epitope that is at least at least 20%, at least 50%, at least 100%, at least 150%, at least 2-fold, at least 2.5 fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 4.5 fold, least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, least 1000-fold, at least 5000-fold, at least 10,000- fold, or more, greater than the binding affinity for the same target epitope of an ABU generated from the reference light chain amino acid sequence and the second heavy chain.

[0031] In some embodiments, the disclosure provides for an ABM comprising at least two ABUs, wherein the first ABU comprises a variant or mutated light chain and a first heavy chain, and a second ABU comprises a second heavy chain and a variant or mutated light chain having the same amino acid sequence as the variant or mutated light chain m the first ABU. In some embodiments, the first ABU is associated with a binding affinity for a target epitope that is at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 2-fold, at least 2.5 fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 4.5 fold, least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900- fold, least 1000-fold, at least 5000-fold, at least 10, 000-fold, or more, greater than the binding affinity for the same target epitope of an ABU generated from a reference light chain amino acid sequence and the first heavy chain; and the second ABU is associated with a binding affinity for a target epitope that is at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 2-fold, at least 2.5 fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 4.5 fold, least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250- fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, least 1000-fold, at least 5000-fold, at least 10,000-fold, or more, greater than the binding affinity for the same target epitope of an ABU generated from a reference light chain ammo acid sequence and the second heavy chain. In some embodiments, the first ABU is associated with a binding affinity for a target epitope that is at least 10% at least 20%, at least 50%, at least 100%, at least 150%, at least 2-fold, at least 2.5 fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 4.5 fold, least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, least 1000-fold, at least 5000-fold, at least 10, 000-fold, or more, greater than the binding affinity for the same target epitope of an ABU generated from a reference light chain ammo acid sequence and the first heavy chain; and the second ABU is associated with a binding affinity for a target epitope that is not less than 200%, not less than 150%, not less than 100%, not less than 75%, not less than 50%, not less than 25%, or not less than 10% of the binding affinity for the same target epitope of an ABU generated from a reference light chain amino acid sequence and the second heavy chain.

[0032] In some embodiments, the light chain is human. In some embodiments, the light chain is humanized. In some embodiments, the light chain is chimeric. In some embodiments, the light chain comprises a constant domain (CL). In some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda light chain. In some

embodiments, the lambda light chain is a lΐ, l2, l3, or l4 light chain. In some embodiments, the light chain does not include a constant domain. In some embodiments, the light chain is a variable light domain.

[0033] In some embodiments, the light chain (e.g., any of the variant or mutated light chains disclosed herein) is identified as being compatible with one or more heavy chains using any of the methods disclosed herein. In some embodiments, the disclosure provides for a Database comprising amino acid sequence information for one or more light chains. In some

embodiments, the disclosure provides for a method of identifying a light chain that is compatible with one or more heavy chains. In some embodiments, once a variant or mutated light chain identified using any of the methods disclosed herein is paired with a heavy chain, the CDRs of the variant or mutated light chain are not further optimized. In some embodiments, once a variant or mutated light chain identified using any of the methods disclosed herein is paired with a heavy chain, the framework regions of the variant or mutated light chain are not further optimized. In some embodiments, once a variant or mutated light chain identified using any of the methods disclosed herein is paired with a heavy chain, the CDRs of the variant or mutated light chain and of the heavy chain are not further optimized. In some embodiments, once a variant or mutated light chain identified using any of the methods disclosed herein is paired with a heavy chain, the CDRs and the framework regions of the variant or mutated light chain and of the heavy chain are not further optimized. In some embodiments, once a variant or mutated light chain identified using any of the methods disclosed herein is paired with a heavy chain, the constant domain of the variant or mutated light chain (if the mutated light comprises a constant domain) is further optimized.

[0034] In some embodiments, the disclosure provides for a library comprising a plurality' of any of the light chains disclosed herein. In some embodiments, the library comprising a plurality' of light chains is characterized using one or more steps typically involved in deep mutational scanning, including, (i) making a site saturation library', (li) panning and/or sorting of the site saturation library, (iii) next generation sequencing analysis of the panned and/or sorted site saturation library', and/or (iv) calculation of enrichment ratios. In some embodiments, the library is generated, in part, by utilizing single site saturation mutagenesis of a reference light chain amino acid sequence. In some embodiments, the single site saturation mutagenesis is performed by introducing single degenerate codons at each position within a predesignated portion (e.g., a CDR or portion thereof) of the reference light chain amino acid sequence. In some

embodiments, the single site saturation mutagenesis is performed by introducing single degenerate codons at each CDRI , CDR2, and/or CDR3 position within the reference light chain amino acid sequence. In some embodiments, the single site saturation mutagenesis is performed by introducing single degenerate codons at each Framework 1 , Framework 2, Framework 3 and/or Framework 4 position within the reference light chain amino acid sequence. In some embodiments, the degenerate codons are NNK degenerate codons (N=G, A, T or C; K= G or T). In some embodiments, the library comprises a plurality of different variant or mutated light chains, wherein each variant or mutated light chain differs from any other variant or mutated light chain in the library by no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 ammo acids. In some embodiments, the library comprises a plurality of different variant or mutated light chains, wherein each variant or mutated light chain differs from any' other variant or mutated light chain in the library by no more than 1 amino acids. In some embodiments, the no more than 1 amino acid difference between one variant or mutated light chain in the library and another variant or mutated light chain in the library is in a CDR of the light chains. In some embodiments, the no more than 1 amino acid difference between one variant or mutated light chain in the library and another variant or mutated light chain in the librar is in a framework region of the light chains. In some embodiments, the library comprises a plurality of different variant or mutated light chains, wherein each variant or mutated light chain differs from a reference light chain ammo acid sequence by no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acids. In some embodiments, the library comprises a plurality of different variant or mutated light chains, wherein each variant or mutated light chain differs from a reference light chain ammo acid sequence by no more than 1 ammo acids. In some embodiments, the no more than 1 amino acid difference between one variant or mutated light chain in the library and a reference light chain ammo acid sequence is in a CDR of the light chain. In some embodiments, the no more than 1 amino acid difference between one variant or mutated light chain in the library and a reference light chain amino acid sequence is in a framework region of the light chain.

Heavy Chains

[0035] In some embodiments, the disclosure provides for an ABU comprising any of the variant or mutated light chains disclosed herein and a heavy chain. In some embodiments, the heavy chain comprises three CDRs. In some embodiments, the heavy chain has been mutated as compared to a reference heavy chain amino acid sequence. In some embodiments, a heavy chain in an ABU has been affinity matured to improve/optimize binding of the ABU for a specific epitope.

[0036] In some embodiments, the heavy chain is human. In some embodiments, the heavy chain is humanized. In some embodiments, the heavy chain is chimeric. In some embodiments, the heavy chain comprises a variable heavy domain. In some embodiments, the heavy chain comprises an Fc domain. In some embodiments, the heavy chain comprises a CHI, CH2, and/or CH3 domain. In some embodiments, the heavy chain comprises one or more mutations in the Fc domain. In some embodiments, the heavy chain comprises one or more annno acid mutations that, e.g., promote heteroinultimerization (e.g., heterodimerization) with other heavy chains, promote serum half-life, and/or modify effector function. In some embodiments, the mutation is present in a CH3 domain of the heavy chain. (See, e.g., Xu et al. (2015) mAbs 7(1): 231-42.).

[0037] In some embodiments, the ABMs or ABUs described herein comprise an altered heavy chain constant region that has reduced (or no) effector function relative to its

corresponding unaltered constant region. Effector functions involving the constant region of the ABMs or ABUs described herein may be modulated by altering properties of the constant or Fc region. Altered effector functions include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc-receptors, and pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject ABU or ABM containing an altered constant region as compared to the activity of the unaltered form of the constant region. In particular embodiments, modulation includes situations in which an activity is abolished or completely absent.

[0038] In some embodiments, once a heavy chain is paired with a variant or mutated light chain identified using any of the methods disclosed herein, the CDRs of the heavy chain are not further optimized. In some embodiments, once a heavy chain is paired with a variant or mutated light chain identified using any of the methods disclosed herein, the framework regions of the heavy chain are not further optimized. In some embodiments, once a heavy chain is paired with a variant or mutated light chain identified using any of the methods disclosed herein, the CDRs of the variant or mutated light chain and of the heavy chain are not further optimized. In some embodiments, once a heavy chain is paired with a variant or mutated light chain identified using any of the methods disclosed herein, the constant domain(s) of the heavy chain (if the heavy- chain comprises constant domain(s)) are further optimized. In some embodiments, the constant domain(s) of the heavy chain are further altered in the process of isotype switching. In some embodiments, the constant domain(s) of the heavy chain are further altered to facilitate“knob into holes” amino acid alterations.

Antigen-Binding Units and Antigen-Binding Molecules

[0039] In some embodiments, the disclosure provides for an antigen-binding unit (ABU) comprising a light (e.g , a variant or mutated light chain) and a heavy chain. In some embodiments, the disclosure provides for multispecific antigen-binding molecules (ABMs) comprising two or more ABUs. In some embodiments, the disclosure provides for multispecific ABMs comprising two or more ABUs, wherein the light chain amino acid sequence for each ABU is the same (i.e., a common light chain), and wherein the heavy chain amino acid sequence for each ABU is not the same. In some embodiments, the light chain amino acid sequence (e.g., a common/compatible light chain) for use in such ABMs is identified using any of the methods disclosed herein. As used herein, the terms ABU and ABM comprise any of the antibodies, antigen-binding fragments, and/or non-antibody scaffold proteins disclosed herein.

[0040] In some embodiments, any of the multispecific ABMs disclosed herein bind to two or more different epitopes on two or more different targets (e.g., on two or more different proteins). In some embodiments, any of the multispecific ABMs disclosed herein bind to two or more different epitopes on the same target (e.g., on the same protein).

[0041] One skilled in the art wall recognize that other fragments or the full-length protein can also be used as a target for the multispecific ABM disclosed herein. In one example, an antigen of interest is an " antigenic fragment" of a full-length antigen sequence. An "antigenic fragment” refers to a portion of a protein which, when presented by a cell m the context of a molecule of the MHC, can in a T-cell activation assay, activate a T-cell against a cell expressing the protein. Typically, such fragments that bind to MHC class I molecules are 8 to 12 contiguous amino acids of a full length antigen, although longer fragments may also be used. In some examples, the antigenic fragment is one that can specifically bind to an MHC molecule on the surface of an antigen presenting cell (APC), with out further processing of the epitope sequence. In particular examples, the antigenic fragment is 8-50 contiguous amino acids from a full-length antigen sequence, such as 8-20 amino acids, 8-15 amino acids, 8-12 amino acids, 8-10 amino acids, or 8, 9, 10, 1 1 , 12, 13, 14, 15 or 20 contiguous amino acids from a full-length antigen sequence.

[0042] As used herein, an antigen-binding unit (ABU) refers to a domain, region, or the like, of that binds to an antigen. As used herein, a multispecific ABM comprises two or more ABUs. A first ABU forms a separate binding area of the multispecific ABM from a second ABU of the ABM, each unit forming a separate region of antigen binding. Generally, one ABU (first ABU) is distinct from the other ABU (second ABU) in its antigen binding. For example, one ABU of the antibody is monovalent for and binds to one antigen or epitope while the other antigen binding unit of the antibody is monovalent for and binds to a different antigen or epitope.

[0043] The term valency, when used to describe an ABM, refers to the number of recognition (binding) sites in the ABM. Each recognition site specifically recognizes, and is therefore capable of binding, one epitope (binding site) on an antigen. When an ABM comprises more than one recognition site (e.g., when an ABM is an IgG, which has two recognition sites m its variable regions), each recognition site can specifically recognize the same epitope on the same antigen, or different epitopes, whether on the same or different antigens.

[0044] With regard to the binding of an ABU to a target molecule, the terms“specific binding,”“specifically binds to,”“specific for,”“selectively binds,”“selective for,” and the like as related to a particular target antigen or molecule (e.g., a polypeptide target) or an epitope on a particular target antigen or molecule, mean binding that is measurably different from a non specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a target molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.

[0045] The term epitope, as used herein, means a component of an antigen capable of specific binding to an ABM or ABU. Epitopes frequently consist of surface-accessible amino acid residues and/or sugar side chains and can have specific three-dimensional structural

characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost m the presence of denaturing solvents. An epitope can comprise amino acid residues that are directly involved m the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antigen binding protein binds can be determined using known techniques for epitope determination such as, for example, testing for antigen binding protein binding to antigen variants with different point mutations, and/or X-ray cry stall ography techniques.

[0046] In some embodiments, at least one ABU in an ABM has a KD of at least 1 x 10 ⁷ M, at least 1 x 10 ⁸ M, at least 1 x 10 ⁹ M, at least 1 x lO ¹⁰ M, at least 1 x 10 ¹¹ M, at least 1 x 10 ¹² M, or at least 1 x 10 ¹³ M. In some embodiments, more than one ABUs in an ABM have the same or similar KD.

[0047] The term KD (M), as used herein, refers to the equilibrium dissociation constant of a particular antigen binding unit /antigen interaction. KD = ky/ka. The term kd (sec ¹), as used herein, refers to the dissociation rate constant of a particular antigen binding unit/antigen interaction. This value is also referred to as the koir value. The term k_a (M^_1 sec A as used herein, refers to the association rate constant of a particular antigen binding unit/antigen interaction. This value is also referred to as the k_0n value.

[0048] In some embodiments, the binding of one ABU (e.g., the first ABU) of the

multispecific ABM to its target does not block or stencally hinder the binding of the other ABU (e.g., the second ABU) to its target. For example, upon the binding of a first ABU to a first antigen, the second ABU is free to bind a second antigen. Thus, in some embodiments, the first ABU and second ABU bind to their respective targets concurrently.

[0049] In some embodiments, binding of the first ABU and the second ABU to their respective targets bridges an immune cell and a second cell (e.g., a cancer cell or microbial cell) together, bringing the two cells in close proximity. As used herein, bridge refers to the joining of two cell types or bringing of the two cells together in close proximity, such that the two cells need not be in physical contact. Thus, the multispecific ABM acts as a connector (e.g., a bridge) to the two cells.

[0050] Methods for determining whether two cells are bridged by an ABM of the present disclosure are known in the art. For example, in some embodiments, the bridging of the immune cell and the second cell is determined by, e.g., flow cytometry, FRET, immunoprecipitation, microscopy, or fluorescence plate reader.

[0051] As described herein, the disclosed multi specific ABMs include bispecific, trispecific, tetraspecific, or further multispecific antibodies or antigen binding fragments thereof.

[0052] The term immunoglobulin or antibody, as used herein, refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (I.) chains and one pair of heavy (H) chains. In an intact immunoglobulin, all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well

characterized. See, e.g, Paul (2013) Fundamental Immunology 7th ed., Ch. 5, Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy⁷ chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH).

[0053] An antibody, as used herein, can refer to intact antibodies (e.g., intact

immunoglobulins) and antibody fragments. However, antigen binding fragments can be used interchangeably with an intact antibody. Antigen binding fragments comprise at least one antigen binding domain. One example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer. Antibodies and antigen binding fragments can be described by the antigen to which they specifically bind.

[0054] The VH and VL regions can be further subdivided into regions of hypervariability (hypervariable regions (HVRs), also called complementarity determining regions (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding, and confer antigen specificity and binding affinity to the ABU (e.g., antibody). (See Rabat et al. (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD.)

[0055] The term chimeric antibody refers to an antibody m which a component of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

[0056] Humanized forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies can also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. (See Jones et al. (1986) Nature , 321 :522-525; Riechmann et al. (1988) Nature, 332:323-329; and Presta, (1992) Curr Op Struct Biol, 2:593-596).

[0057] A human antibody is one that possesses an ammo acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g, obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0058] In some embodiments, an antibody molecule comprises a diabody, and a single-chain molecule, as well as an antigen binding fragment of an antibody (e.g., Fab, F(ab')2, and Fv). For example, an antibody molecule can include a heavy (H) chain variable domain sequence

(abbreviated herein as UΉ), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab', F(ab')?., Fc, Fd, Fd', Fv, single chain antibodies (scFv, for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, XgD, and IgE, and from any subclass (e.g., IgGl, XgG2, IgG3, and IgG4) of antibodies. The preparation of antibody molecules can be monoclonal or polyclonal. An antibody molecule can also be a human, humanized, CDR-grafted, or an in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl , IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from either kappa or lambda light chains.

[0059] Antigen binding fragments of an antibody molecule are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0060] Antibody molecules can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, hut are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies are disclosed in WO 94/04678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Came!idae species (e.g., camel, llama, dromedary, alpaca and guanaco) or other species besides Camelidae.

[0061] In some embodiments, an ABU can also be or can also comprise, e.g., a non-antibody, scaffold protein. These proteins are, generally, obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins. For exampl e, the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali et al. (1999) ,/. Biol. Cheni. 274:24066-24073. The portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites. The libraries are then screened, as an antibody library is, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimal selectivity and affinity for the target antigen. See, e.g.. Hey et al (2005) TRENDS Biotechnol 23(10): 514-522.

[0062] One of skill in the art would appreciate that the scaffold portion of the non-antibody scaffold protein can include, e.g, all or part of the Z domain of S. aureus protein A, human transferrin, human tenth fibronectm type III domain, kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human lipocalin (e.g., anticalins, such as those described in, e.g, WO2015/104406), human crystallin, human ubiquitin a trypsin inhibitor from E. elaterium, or a Variable Lymphocyte Receptor (see, e.g., Boehm et al, 2012, Annu. Rev. Immunol., 30:203-20).

[0063] Various multispecific antibody formats are known in the art, including, for example, a multispecific IgG, a multispecific antibody fragment, a multispecific fusion protein, an appended IgG, and a multispecific antibody conjugate, described herein. Exemplary multispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv- based or diabody multispecific formats, IgG-scFv fusions, dual variable domain (DVD)-ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgGl /lgG2, dual acting Fab (DAF)-lgG, and Mab² multispecific formats (see, e.g., Klein et al. (2012) niAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). (See also Spiess et al. (2015) Mol Immunol 67:95-106.) Multispecific antibodies can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self- assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al. (2013) J Am Chem Soc 135 ( 1 ) : 340-6).

[0064] Methods for generating multispecific antibody molecules are known in the art, including but not limited to, for example, the“knob into hole” approach described in, e.g., U.S. Pat. No. 5,731,168; the electrostatic steering Fc pairing as described in, e.g., WO 09/089004,

WO 06/106905 and WO 2010/129304; Strand Exchange Engineered Domains (SEED) heterodimer formation as described in, e.g., WO 07/1 10205; Fab arm exchange as described in, e.g., WO 08/1 19353, WO 201 1 /131746, and WO 2013/060867; double antibody conjugate, e.g, by antibody cross-linking to generate a multispecific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryi reactive group as described in, e.g, U.S. Pat.

No. 4,433,059; multispecific antibody determinants generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycle of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g, U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g, three Fab' fragments cross-linked through sulfhdryl reactive groups, as described in, e.g, U.S. Pat. No. 5,273,743; biosynthetic binding proteins, e.g., pair of scFvs cross-linked through C-terminal tails preferably through disulfide or amine-reactive chemical cross-linking, as described in, e.g., U.S. Pat. No. 5,534,254; multifunctional antibodies, e.g., Fab fragments with different binding specificities multimerized through leucine zippers (e.g., c-fos and c-jun) that have replaced the constant domain, as described in, e.g., U.S. Pat. No. 5,582,996; bispecific and ohgospecific mono- and oligo-valent receptors, e.g., VH-CHl regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CHI region of one antibody and the VH region of the other antibody typically with associated light chains, as described in, e.g, U.S. Pat. No. 5,591 ,828; bispecific DNA-antibody conjugates, e.g, cross!inking of antibodies or Fab fragments through a double stranded piece of DNA, as described in, e.g, U.S. Pat. No. 5,635,602; multispecific fusion proteins, e.g, an expression construct containing two or more scFvs with a hydrophilic helical peptide linker between them and a full constant region, as described in, e.g., U.S. Pat. No. 5,637,481; multivalent and multispecific binding proteins, e.g., dimer of polypeptides having first domain with binding region of Ig heavy chain variable region, and second domain with binding region of Ig light chain variable region, generally termed diabodies (higher order structures are also encompassed creating for bispecific, trispecific, or tetraspecifie molecules, as described in, e.g., U.S. Pat. No. 5,837,242; mimbody constructs with linked VL and VH chains further connected with peptide spacers to an antibody hinge region and CHS region, which can be dimerized to form

bispeeific/rnu!tivalent molecules, as described in, e.g., U.S. Pat. No. 5,837,821; VH and VL domains linked with a short peptide linker (e.g., 5 or 10 amino acids) or no linker at all in either orientation, which can form dimers to form bispecific diabodies; trimers and tetramers, as described in, e.g., U.S. Pat No. 5,844,094; String ofVH domains (or VL domains in family members) connected by peptide linkages with crosslinkable groups at the C-terminus further associated with VL domains to form a senes of FVs (or scFvs), as described in, e.g., U.S. Pat No. 5,864,019; and single chain binding polypeptides with both a VH and a VL domain linked through a peptide linker are combined into multivalent structures through non-covalent or chemical crosslinking to form, e.g., homobivalent, heterobivalent, tri valent, and tetravalent structures using both scFV or diabody type format, as described in, e.g., U.S. Pat No. 5,869,620. Additional exemplary multispecific and bispecific ABMs and methods of making the same are found, for example, in U.S. Pat. Nos. 5,910,573; 5,932,448; 5,959,083; 5,989,830; 6,005,079;

6,239,259; 6,294,353; 6,333,396; 6,476,198; 6,51 1 ,663; 6,670,453; 6,743,896; 6,809,185;

6,833,441 ; 7,129,330; 7,183,076; 7,521 ,056; 7,527,787; 7,534,866; 7,612,181; US Patent

Publication Nos. US2Q02004587A1, US2002076406A1, US2002103345A1 , US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1, US2005003403A1, US2005004352A1, US2005069552A1 , US2005079170A1,

U S2005100543 A 1 , US2005136049A1, US200513605I Al, US2005163782A1,

US2QQ5266425A1, US2006083747A1, US2006120960A1 , US2006204493A1,

US2006263367A1, US2007004909A1, US200708738I Al, US2007128150A1,

US2007141049A1 , US2007154901 Al, US2007274985A1 , US2008050370A1,

U S2008069820 A 1 , US2008152645A1, US2008171855A1, US2008241884A1,

US2008254512A1 , US2008260738A1, US2009130106A1, US2009148905A1,

US2009155275 Al , US2009162359A1, US2009162360A1, US2009175851A1,

US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1,

US2009274649A1 ; European Patent No. EP346087A2; and PCX Publication Nos.

W00006605A2, WO02072635A2, W004081051A1, WQ06020258A2, W02007044887A2, W02007095338 A2, WQ2QQ7137760A2, WQ2008119353A1, W02009021754A2,

W02009068630A1 , WO9103493A1, W09323537A1, WO9409131A1, W09412625A2,

WO9509917A1 , W09637621 A2, WO9964460A1.

[0065] In some embodiments, the first ABU or second ABU (or any further ABUs), or both (or all), can comprise a heavy chain comprising one or more immunoglobulin Fc modifications. In some embodiments, the immunoglobulin Fc domain of the heavy chain comprises one or more amino acid mutations that, e.g., promote heteromu!timerization (e.g., heterodimerization) of the first and second ABU, promote serum half-life, and/or modify effector function. In some embodiments, the mutation is present in a CH3 domain of the heavy chain. (See, e.g., Xu et al. (2015) mAbs 7(1): 231 -42.)

[0066] While traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs, a variety of engineered Fc domains have been designed as asymmetric interaction pairs (Spiess et al. (2015 ) Molecular Immunology 67(2A); 95-106) to promote heter omul tirn erization (e.g., heterodimerization), e.g., of a first ABU and a second ABU. Various methods are known m the art that increase desired pairing of Fc-containing polypeptide chains in a single cell line to produce a preferred asymmetric fusion protein at acceptable yields (see, for example, Klein et al. (2012) mAbs 4:653-663; and Spiess et al. (2015) Molecular Immunology 67(2PartA): 95-106). Methods to obtain desired pairing of Fc-containing polypeptides include, but are not limited to, charge-based pairing (electrostatic steering),“knobs-into-holes” steric pairing, SEEDbody pairing, and leucine zipper-based pairing. (See, for example, Ridgway et al. (1996) Protein Eng 9:617-621 ; Merchant et al. (1998) Nat Biotech 16:677-681 ; Davis et al. (2010) Protein EngDes Sel 23: 195-202; Gunasekaran et al. (2010) J Biol Chem 285: 19637- 19646; Wramk et al. (2012) J Biol Chem 287:43331 -43339; US Patent No. 5932448; and PCX Publication Nos. WO 1993/011162; WO 2009/089004, and WO 2011/034605.

[0067] For example, one means by which interaction between specific polypeptides may be promoted is by engineering protuberance-into-cavity (knob-into-holes) complementary regions such as described m U.S. Patent Nos. 7,183,076 and 5,731,168; and PCX Publication No. WO 2016/164089. “Protuberances” are constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., a first interaction pair) with larger side chains (e.g, tyrosine or tryptophan). Complementary“cavities” of identical or similar size to the

protuberances are optionally created on the interface of the second polypeptide (e.g., a second interaction pair) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Where a suitably positioned and dimensioned protuberance or cavity exists at the interface of either the first or second polypeptide, it is only necessary' to engineer a

corresponding cavity'^· or protuberance, respectively, at the adjacent interface.

[0068] At neutral pH (7.0), aspartic acid and glutamic acid are negatively charged and lysine, arginine, and histidine are positively charged. These charged residues can be used to promote heterodimer formation and at the same time hinder homodimer formation. Attractive interactions take place between opposite charges and repulsive interactions occur between like charges. In part, protein complexes disclosed herein make use of the attractive interactions for promoting heteromul timer formation (e.g., heterodimer formation), and optionally repulsive interactions for hindering homomultimer formation (e.g., homodimer formation) by carrying out site directed mutagenesis of charged interface residues.

[0069] For example, the IgGl CHS domain interface comprises four unique charge residue pairs involved in domain-domain interactions: Asp356-Lys439', Glu357-Lys370', Lys392~ Asp399', and Asp399-Lys409' [residue numbering in the second chain is indicated by (')]. It should be noted that the numbering scheme used here to designate residues in the IgGl CHS domain conforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetry present in the CH3-CH3 domain interactions, each unique interaction is represented twice in the structure (e.g., Asp-399-Lys409' and Lys409-Asp399'). In the wild-type sequence, K409-D399' favors both heterodimer and homodimer formation. A single mutation switching the charge polarity (e.g., K409E; positive to negative charge) m the first chain leads to unfavorable interactions for the formation of the first chain homodimer. The unfavorable interactions arise due to the repulsive interactions occurring between the same charges (negative-negative; K409E-D399' and D399-K409E’). A similar mutation switching the charge polarity⁷ (D399K'; negative to positive) m the second chain leads to unfavorable interactions (K409'-D399K^f and D399K-K409') for the second chain homodimer formation. But, at the same time, these two mutations (K409E and D399K') lead to favorable interactions (K409E-D399K' and D399-K409') for the heterodimer formation. The electrostatic steering effect on heterodimer formation and homodimer discouragement can be further enhanced by mutation of additional charge residues which may or may not be paired with an oppositely charged residue in the second chain including, for example, Arg355 and Lys360. (See, e.g., PCT Publication No. WO 2016/164089.)

[0070] Thus, in some embodiments, the multispecific ABMs described herein may comprise a constant domain of an immunoglobulin, including, for example, the Fc portion of an

immunoglobulin. For example, a first ABU may comprise an ammo acid sequence that is derived from an Fc domain of an IgG (IgGl , XgG2, IgG3, or IgG4), IgA (IgA! or IgA2), IgE, or XgM immunoglobulin. Optionally, a second (or further) ABU may comprise an amino acid sequence that is derived from an Fc domain of an IgG (IgG!, lgG2, !gG3, or XgG4), IgA (IgAl or XgA:2), IgE, or XgM. Such immunoglobulin domains may comprise one or more amino acid modifications (e.g., deletions, additions, and/or substitutions) that promote heteromultimer (e.g., heterodimer) formation. In some embodiments, a first ABU and a second ABU comprise Fc domains derived from the same immunoglobulin class and subtype. In some embodiments, a first and second ABU comprise Fc domains derived from different immunoglobulin classes or subtypes. Similarly, a first and/or a second ABU (e.g., an asymmetric pair or an unguided interaction pair) comprise a modified constant domain of an immunoglobulin, including, for example, one or more amino acid modifications (e.g., deletions, additions, and/or substitutions) that promote heteromultimer (e.g., heterodimer) formation. Methods of generating Fc modifications having the desired heterodimer formation are known in the art.

[0071] In some embodiments, the Fc domain can be modified to enhance serum half-life of the multispecific ABM disclosed herein. Fc domains comprising one or more mutations which enhance or diminish antibody binding to the Fc receptor, e.g., at acidic pH as compared to neutral pH, are known in the art. For example, the ABMs or ABUs disclosed herein may comprise a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g:, m an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the ABM or ABU when administered to an animal. Methods of modifying the Fc domain for desired characteristics, such as enhanced serum half-life are known in the art.

[0072] In some embodiments, the ABMs or ABUs described herein comprise an altered heavy chain constant region that has reduced (or no) effector function relative to its

corresponding unaltered constant region. Effector functions involving the constant region of the ABMs or ABUs described herein may be modulated by altering properties of the constant or Fc region. Altered effector functions include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc-receptors, and pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity' exhibited by a subject ABM or ABU containing an altered constant region as compared to the activity of the unaltered form of the constant region. In particular embodiments, modulation includes situations in which an activity is abolished or completely absent.

[0073] An altered constant region with altered FcR binding affinity and/or ADCC activity and/or altered CDC activity is a polypeptide that has either an enhanced or diminished FcR binding activity and/or ADCC activity and/or CDC activity compared to the unaltered form of the constant region. An altered constant region that displays increased binding to an FcR binds at least one FcR with greater affinity than the unaltered polypeptide. An altered constant region which displays decreased binding to an FcR binds at least one FcR with lower affinity than the unaltered form of the constant region. Such variants that display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the binding to the FcR as compared to the level of binding of a native sequence immunoglobulin constant or Fc region to the FcR. Similarly, an altered constant region that displays modulated ADCC and/or CDC activity may exhibit either increased or reduced ADCC and/or CDC activity compared to the unaltered constant region. For example, in some embodiments, an ABU or ABM comprises an altered constant region can exhibit approximately 0 to 50% (e.g., less than 50, 49, 48, 47, 46,

45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20,

19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the ADCC and/or CDC activity of the unaltered form of the constant region. An ABU or multispecific ABM described herein comprising an altered constant region displaying reduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDC activity.

[0074] In some embodiments, the multispecific ABMs described herein exhibit reduced or no effector function. In some embodiments, the multispecific ABM comprises a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region. (See e.g . Burton et al. (1992) Advlmmun 51 : 1-18; Canfield et al. (1991) J Exp Med 173: 1483-1491; and Mueller et al. (1997) Mo! Immunol 34(6):441-452).

[0075] In some embodiments, the multispecific ABMs contain an altered constant region exhibiting enhanced or reduced complement dependent cytotoxicity (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fc region of the antibody. See, e.g , U.S. Patent No. 6,194,551.

[0076] ABMs or ABUs can be further selected for binding to more than one species. For example, antibodies or fragments that bind both mouse and human can be selected by screening with both mouse and human target cells.

IP. DATABASE

[0077] In some embodiments, the disclosure provides for a Database comprising amino acid sequence information for any two or more of the light chains disclosed herein. The term “Database,” as used herein, refers to a stored collection of information relating to a plurality of light chain ammo acid sequences, wherein the light chain amino acid sequences comprise one or more amino acid alterations (e.g. , substitutions, insertions, deletions) as compared to a single reference light chain amino acid sequence. In some embodiments, the Database is a stored collection of information relating to a plurality of light chain ammo acid sequences, wherein a subset of the light chain ammo acid sequences comprise one or more ammo acid alterations (e.g., substitutions, insertions, deletions) as compared to a single reference light chain ammo acid sequence. Such altered light chain amino acid sequences are referred to herein as variant or mutated light chain amino acid sequences. Reference light chain amino acid sequences include, for example, human germline light chain amino acid sequences. In some embodiments, the stored collection of information includes amino acid sequence information, and, for example, corresponding nucleic acid sequence information of at least a portion of the variant or mutated light chains (e.g., complementarity determining regions, framework regions, and/or constant domain regions). In some embodiments, the stored collection of information includes, for example, binding affinity, species cross-reactivity, target specificity, and/or half-life information of an antigen-binding unit (ABU) comprising any of the variant or mutated light chain amino acid sequences stored in the Database and a heavy chain. In some embodiments, the disclosure provides for a database comprising: a) a plurality of mutated light chain amino acid sequences; b) a plurality of heavy chain amino acid sequences: and c) binding affinity' data of a plurality of ABUs for one or more antigens or epitopes, wherein each of the ABUs comprises a mutated light chain amino acid sequence selected from a) and a heavy chain amino acid sequence selected from b).

[0078] In particular embodiments, the Database further comprises amino acid sequence information for at least a portion of one or more heavy chain amino acid sequences (e.g., complementarity determining regions, framework regions, and/or constant domain regions). In some embodiments, the Database comprises amino acid sequence information for one or more ABUs comprising a light chain amino acid sequence and a heavy chain ammo acid sequence. In some embodiments, the Database comprises information relating to characteristics (e.g., binding affinity data) associated with one or more ABUs.

[0079] In some embodiments, the Database comprises one or more light chain amino acid sequences. In some embodiments, the Database comprises, 5 or more, 10 or more, 20 or more,

30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, 4500 or more, or 5000 or more light chain amino acid sequences. In some embodiments, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more,

550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more,

900 or more, 950 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, 4500 or more, or 5000 of the light chain ammo acid sequences have been altered or mutated in comparison to a single reference light chain amino acid sequence, such as a human germline light chain ammo acid sequence. In some

embodiments, the variant or mutated light chain amino acid sequence comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 15, or no more than 20 amino acid alterations (e.g., amino acid substitutions, insertions or deletions) as compared to a single reference light chain ammo acid sequence. In some embodiments, the amino acid alterations occur in the variable region of the single reference light chain amino acid sequence. In some embodiments, the amino acid alterations occur in one or more CDRs of the single reference light chain amino acid sequence. In some embodiments, the variant or mutated light chain amino acid sequence comprises no more than 1 amino acid alteration (e.g., amino acid substitutions, insertions or deletions) as compared to a single reference light chain amino acid sequence. In some embodiments, the no more than 1 amino acid alteration (e.g., amino acid substitution, insertion or deletion) occurs in the variable region of the single reference light chain amino acid sequence. In some embodiments, the no more than 1 amino acid alteration (e.g., amino acid substitution, insertion or deletion) occurs in a CDR of the single reference light chain amino acid sequence. In some embodiments, the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ammo acid sequences of any one of SEQ ID NOs: 1-4. In some embodiments, the reference light chain amino acid sequence comprises the ammo acid sequence of SEQ ID NO: 1. In some embodiments, the reference light chain amino acid sequence comprises the ammo acid sequence of SEQ ID NO: 2. In some embodiments, the reference light chain amino acid sequence comprises the ammo acid sequence of SEQ ID NO: 3 In some embodiments, the reference light chain ammo acid sequence comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 5-7. In some embodiments, the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NQs: 8-10. In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NQs: 11-13. In some embodiments, the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NQs: 14-16. In some embodiments, the reference light chain ammo acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NQs: 17-19.

[0080] In some embodiments, the Database comprises one or more heavy chain amino acid sequences. In some embodiments, the Database comprises amino acid sequence information for one or more ABU. In some embodiments, the Database comprises amino acid sequence information for a plurality of ABUs in an ABU set, wherein the ABU set comprises 2 or more ABUs each comprising the same heavy chain amino acid sequence, but each comprising a different light chain amino acid sequence. In some embodiments, the Database comprises amino acid sequence information for a plurality of ABUs in an ABU set, wherein the ABU set comprises 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, or 1000 or more ABUs having the same heavy chain amino acid sequence, but a different light chain amino acid sequence. In some embodiments, 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, or 1000 or more of the light chain amino acid sequences m each of the ABU s in the ABU sets have ammo acid sequences that differ from each other by no more than 1 , 2, 3, 4, 5, 10, 15, or 20 ammo acids. In some embodiments, the light chain ammo acid sequences in each of the ABU s in the ABU sets have light chain variable region ammo acid sequences that differ from each other by no more than 1 , 2, 3, 4, 5, 10, 15, or 20 ammo acids. In some embodiments, the light chain amino acid sequences in each of the ABUs in the ABU sets have light chain CDRs that differ from each other by no more than 1, 2, 3, 4, 5, 10, 15, or 20 amino acids. In some embodiments, 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, or 1000 or more of the light chain ammo acid sequences in each of the ABUs in the ABU sets have amino acid sequences that differ from each other by no more than 1 amino acid. In some embodiments, the light chain amino acid sequences in each of the ABUs in the ABU sets have light chain variable region amino acid sequences that differ from each other by no more than 1 ammo acid. In some embodiments, the light chain amino acid sequences in each of the ABUs in the ABU sets have CDRs that differ from each other by no more than 1 amino acid.

[0081] In some embodiments, the Database comprises ammo acid sequence information from a plurality of ABUs from two or more sets of ABUs. In some embodiments, the heavy chain amino acid sequence for the ABUs in one ABU set differs from the heavy chain amino acid sequence for the ABUs in another ABU set. In some embodiments, the ABUs in one ABU set bind to one epitope, while the ABUs in another ABU set bind to another epitope. In some embodiments, the Database comprises amino acid sequence information for a plurality of ABUs from 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, or 1000 or more different sets of ABUs.

[0082] In some embodiments the Database comprises characteristic information relating to one or more characteristics of one or more light chain, heavy chain, ABU and/or ABU sets. In some embodiments, the one or more characteristics are one or more desired characteristics (e.g., information related to binding affinity). In some embodiments, the characteristic information is characteristic information relating to one or more ABU (and/or the light chain and/or heavy chain present in the ABU). In some embodiments, the ABU characteristic information is information related to binding affinity . In some embodiments, the characteristic information is characteristic information relating to one or more ABUs in an ABU set. In some embodiments, the characteristic information is characteristic information for an ABU from an ABU set and is relative to characteristic information of another ABU from the same ABU set. In some embodiments, the characteristic information is characteristic information for an ABU from an ABU set and is relative to characteristic information of another AB U from a different ABU set.

In some embodiments, the ABU characteristic information is the frequency of the ABU in a plurality of ABUs before and after the plurality of ABUs have been subjected to a sorting step.

In some embodiments, the sorting step is a binding assay f e.g., phage panning for binding affinity to a target epitope). In some embodiments, the ABU characteristic information is an enrichment ratio (ER) measuring the enrichment of an ABU following the sorting step. In some embodiments, the ER is calculated according to the following formula: ER=log2(Fi/Fo) where Fs is the ABU frequency after i rounds of sorting and Fo is the frequency of the same ABU in the starting plurality of ABUs.

[0083] In some embodiments, the characteristic information is light chain characteristic information. In some embodiments, the light chain characteristic information is determined by assessing the ABU characteristic information. For example, an ER value can be determined for a specific ABU in a plurality of ABUs, and if each of the ABUs in the plurality of ABUs have the same heavy chain amino acid sequence, then the ER value of the ABUs can be used as a proxy for the ER value of the different light chain components of the ABUs in the plurality of ABUs.

[0084] In some embodiments, the disclosure provides for a method of creating a Database. In some embodiments, the method comprises: a) providing a library comprising a plurality of different variant or mutated light chains; wherein each variant or mutated light chain in the library comprises at least one amino acid mutation as compared to a reference light chain ammo acid sequence; b) providing a set of heavy chains, wherein each of the heavy chains comprises the same ammo acid sequence; c) separately pairing each of the different mutated light chains with heavy chains from the set of heavy chains, thereby generating a library of ABUs capable of binding an antigen; d) screening the ABUs from the library of ABUs for a desired characteristic, e) optionally repeating steps c) and d) with a different set of heavy chains each having an identical amino acid sequence; f) storing in a Database the data obtained from the screening step d) and optionally from the screening step in e). In some embodiments, the data obtained from the screening step d) after performing the method in a first instance is compared with data obtained from the screening step d) after performing the method in a second instance, wherein the variant or mutated light chain amino acid sequence used in the method of the first and second instance was the same, and wherein the set of heavy chains used in the method of the first instance was different that the set of heavy chains used in the second instance. In some embodiments, if the data from the method performed in the first instance indicates that an ABU comprising a specific variant or mutated light chain is associated with a desired characteristic, and if the data from the method performed in the second instance indicates that an ABU comprising the same variant or mutated light chain is associated with a desired characteristic, then the variant or mutated light chain is identified as being compatible with the heavy chain used in the method of the first instance and with the heavy chain used in the method of the second instance.

[0085] In some embodiments, the database is stored in a memory storage device or memory storage system, including, for example, secondary storage devices, removable storage devices, and server and cloud-based storage systems. In some embodiments, the memory storage device is a computer or portabie/mobile device, such as a tablet or smartphone. In some embodiments, the Database is accessible via a computer readable medium. In some embodiments, this computer readable medium has residing thereon machine executable code that when executed by at least one processor, causes the processor to perform steps that include retrieving light chain ammo acid sequence information and/or heavy chain ammo acid sequence information and/or characteristic information associated with an ABU. In some embodiments, the machine executable code further contains instructions in a computer programming language for extracting light chain ammo acid sequence information and/or heavy chain amino acid sequence information and/or characteristic information associated with an ABU. This extracted information can he stored and/or displayed in any format suitable for the user viewing the information (e.g., on a computer display monitor). In some embodiments, the machine executable code further contains instructions in a computer programming language for distinguishing between characteristics associated with an ABU and/or a variant or mutated light chain such that a desired characteristic may he identified. In some embodiments, the Database is stored in a memory storage device in a format suitable for computer automated and/or manual data analysis, and/or for display /printing on a display or printing device linked to a computing system. IV. METHODS OF USE

Methods of Identifying Compatible Light Chains

[0086] In some embodiments, the disclosure provides for methods of identifying a light chain that is compatible with one or more heavy chains. In some embodiments, the light chain is compatible with a heavy chain if an ABU comprising the light chain and the heavy chain is associated with a desired characteristic. In particular embodiments, the desired characteristic is a binding affinity of an ABU for a specific epitope. In some embodiments, the desired

characteristic is an enrichment ratio (ER) of an ABU following a sorting process (e.g., a binding step). In some embodiments, the sorting process is a binding affinity assay. In some

embodiments, the desired characteristic of an ABU is attributable to the light chain component of the ABU.

[0087] In some embodiments, a method for identifying a light chain that is compatible with one or more heavy chains comprises: a) providing a library⁷ comprising a plurality of different variant or mutated light chains; wherein each variant or mutated light chain in the library comprises at least one amino acid mutation as compared to a reference light chain ammo acid sequence; b) providing a set of heavy chains, wherein each of the heavy chains comprises the same amino acid sequence; c) separately pairing each of the different mutated light chains with heavy chains from the set of heavy chains, thereby generating a library⁷ of antigen-binding units (ABUs) capable of binding an antigen; d) screening the ABUs from the library of ABUs for a desired characteristic. In some embodiments, steps c) and d) are repeated with a different set of heavy chains each having an identical amino acid sequence. In some embodiments, a compatible light chain is identified if: i) when paired with a first heavy chain, is associated with a first desired characteristic, ii) when paired with second heavy chain is associated with a second desired characteristic.

[0088] In some embodiments, any of the methods disclosed herein comprise the step of providing a library comprising a plurality of different variant or mutated light chains; wherein each variant or mutated light chain m the library comprises at least one amino acid alteration (e.g., substitution, insertion, and/or deletion) as compared to a reference light chain amino acid sequence. In some embodiments, the steps of preparing the library comprising a plurality of light chains comprises one or more steps typically involved in deep mutational scanning, including, (i) making a site saturation library , (ii) panning and/or sorting of the site saturation library, (iii) next generation sequencing analysis of the panned and/or sorted site saturation library, and/or (iv) calculation of enrichment ratios. In some embodiments, the library is generated, m part, by utilizing single site saturation mutagenesis of a reference light chain ammo acid sequence. In some embodiments, the single site saturation mutagenesis is performed by- introducing single degenerate codons at each position within the reference light chain ammo acid sequence. In some embodiments, the single site saturation mutagenesis is performed by introducing single degenerate codons at each CDR1, CDR2, and/or CDR3 position within the reference light chain ammo acid sequence. In some embodiments, the single site saturation mutagenesis is performed by introducing single degenerate codons at each Framework 1, Framework 2, Framework 3 and/or Framework 4 position within the reference light chain amino acid sequence. In some embodiments, the degenerate codons are NNK degenerate codons (N=G, A, T or C; K= G or T). In some embodiments, the library comprises a plurality of different variant or mutated light chains, wherein each variant or mutated light chain differs from any other variant or mutated light chain in the library by no more than 1 , no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acids. In some embodiments, the library comprises a plurality of different variant or mutated light chains, wherein each variant or mutated light chain differs from any other variant or mutated light chain in the library by no more than 1 amino acids. In some embodiments, the no more than 1 amino acid difference between one variant or mutated light chain in the library and another variant or mutated light chain in the library is in a CDR of the light chains. In some embodiments, the no more than 1 ammo acid difference between one variant or mutated light chain in the library and another variant or mutated light chain in the library is in a framework region of the light chains. In some embodiments, the library comprises a plurality of different variant or mutated light chains, wherein each variant or mutated light chain differs from a reference light chain amino acid sequence by no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 ammo acids. In some embodiments, the library comprises a plurality of different variant or mutated light chains, wherein each variant or mutated light chain differs from a reference light chain amino acid sequence by no more than 1 amino acids.

In some embodiments, the no more than 1 amino acid difference between one variant or mutated light chain in the library and a reference light chain amino acid sequence is in a CDR of the light cham. In some embodiments, the no more than 1 amino acid difference between one variant or mutated light chain in the library and a reference light chain amino acid sequence is in a framework region of the light chain.

[0089] In some embodiments, any of the methods disclosed herein comprise the step of providing a set of heavy chains. In some embodiments, each of the heavy chains comprises the same amino acid sequence. In some embodiments, the heavy chains have been affinity matured to improve/optimize binding to a target antigen. In some embodiments, the heavy chains have been optimized/improved prior to pairing with the variant or mutated light chain. In some embodiments, the binding affinity of an ABU comprising the heavy chain and a reference light chain amino acid sequence has been determined.

[0090] In some embodiments, any of the methods disclosed herein comprise the step of separately pairing a plurality of different mutated light chains with heavy chains from the set of heavy chains (wherein the heavy chains each comprise the same amino acid sequence), thereby generating a library of ABUs capable of binding an antigen. In some embodiments, the disclosure provides for a plurality' of libraries of ABUs. In some embodiments, the plurality of ABU libraries differ with regard to the heavy chains present in each ABU library. In some embodiments, a plurality' of the variant or mutated light chain amino acid sequences present in one ABU library are also present in another ABU library⁷. In some embodiments, the light chain amino acid sequences present in one ABU library⁷ are also present in another ABU library .

[0091] In some embodiments, the method comprises the step of screening a library of ABUs, wherein each ABU in the library comprises the same heavy chain amino acid sequence, but wherein a plurality of the ABUs in the library comprise different variant or mutated light chain amino acid sequences. In some embodiments, the method comprises the step of screening multiple libraries of ABUs, wherein each library of ABUs differs with regard to the heavy chains present in the library of ABUs. For example, in some embodiments, the method comprises: a) the step of screening a first library of ABUs, wherein each ABU in the first library comprises the same heavy chain ammo acid sequence, but wherein a plurality of the ABUs in the first library comprise different variant or mutated light cham amino acid sequences, and b) the step of screening a second library of ABUs, wherein each ABU m the second library comprises the same heavy chain amino acid sequence, but wherein a plurality of the ABUs in the second library comprise different variant or mutated light chain ammo acid sequences, and wherein the heavy chains in the first library of ABUs are different from the heavy chains in the second library of ABUs, and wherein the variant or mutated light chain amino acid sequences present in the first library of ABUs are also present in the second library of ABUs. In some embodiments, the library of ABUs is a library of ceils expressing the library of ABUs.

[0092] In some embodiments, the screening step comprises the step of sorting ABUs from a library of ABUs. In some embodiments, the sorting step comprises sorting the ABUs based on the presence or absence of a desired characteristic. In some embodiments, the sorting step comprises sorting the ABUs based on a threshold value associated with a desired characteristic. In some embodiments, the sorting step comprises sorting the ABUs based on binding affinity' for a specific epitope. In some embodiments, the sorting step comprises a display step. In some embodiments, the display step comprises the use of a display system, such as phage display, yeast display, ribosome display, bacteria display, and/or mammalian display. By using a display system, functional ABUs are displayed on the surface of the system (e.g., the phage or mammalian cell) that carry the polynucleotide sequences encoding them.

[0093] In some embodiments, the sorting step comprises a phage display/phage panning step. In some embodiments, the bacteriophage used in the phage display is Ml 3 phage, fd filamentous phage, T4 phage, T7 phage, and l phage. In some embodiments, the phage are filamentous phage such as fd and Ml 3. In some embodiments, the bacteria used in the phage panning are Escherichia coli bacterial cells. In some embodiments, the E call bacterial cells are TGI ,

SS320, ER2738, or XL 1 -Blue E. coli cells. In some embodiments, the phage display is used to express an ABU, wherein the ABU is an antibody fragment (e.g., a Fab, Fv, or scFV molecule). In some embodiments, the ABIJ is expressed as a recombinantly-fused protein to any of the phage coat proteins pill, pVIEI, or pIX. (See, e.g., Shi et al. (2010) JMB 397:385-396.) Examples of phage display methods that can be used include those disclosed in Brinkman et al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995 ) J Immunol Methods 184: 177-186;

Kettleborough et al. (1994) Ear J Immunol 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994 ) Advances in Immunology 57: 191-280; and PCX publication nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, and WO 95/20401. Suitable methods are also described in, e.g., US. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571 ,698; 5,427,908; 5,516,637;

5,780,225; 5,658,727; 5,733,743 and 5,969,108. [0094] In some embodiments, the sorting step composes a mammalian cell display step. In some embodiments, the mammalian cell display step comprises transiently transfecting a mammalian cell with one or more polynucleotides encoding any of the ABUs disclosed herein.

In some embodiments, the mammalian ceil display step comprises stably transfecting a mammalian cell with one or more polynucleotides encoding any of the ABUs disclosed herein.

In some embodiments, the mammalian cell display is used to express an ABU, wherein the ABU is an antibody fragment (e.g., a Fab or scFV molecule). In some embodiments, the mammalian ceil display is used to express an ABU, wherein the ABU is a full-length antibody (e.g , a bispecific full-length antibody). Mammalian cell display is particularly useful when trying to preserve post-translational modifications that may be required for ABU function. In some embodiments, the host cells used in the mammalian cell display are HEK cells (e.g., HEK-293 ceils), COS cells, lymphoma- derived B ceils, BHK cells, and/or CHQ cells.

[0095] In some embodiments, the display step is combined with a step of panning/sorting the displayed ABUs for a desired characteristic. In some embodiments, the display step is combined with a step of panning/sorting the displayed ABUs for binding affinity to a specific epitope. In some embodiments, the display step comprises incubating the displayed ABUs on the surface of the display system (e.g., a phage display system or a mammalian display system) with an antigen having a target epitope. In some embodiments, the method further comprises sorting the cells/phage expressing the displayed ABUs based on pre-designated binding threshold affinity values of the displayed ABUs for the epitope. In some embodiments, the cells/phage expressing the displayed ABUs are sorted based on a comparison to the binding affinity of a reference ABU. For example, in some embodiments, the cells/phage expressing the displayed ABUs are sorted if they have a greater binding affinity than a reference ABU In some embodiments, the reference ABU comprises the same heavy chain as the displayed ABUs, but comprises a reference light chain ammo acid sequence (e.g., a reference light chain comprising the amino acid sequence of any one of SEQ ID NOs: 1 -4) rather than a variant or mutated light chain amino acid sequence.

[0096] In some embodiments, following the sorting step, ABU amino acid sequences are determined by sequencing DNA from the display system. In some embodiments, the DNA is sequenced using next generation sequencing. In some embodiments, the method comprises the step of isolating DNA from the display system and amplifying the isolated DNA. In some embodiments, the isolated DNA is ligated to a unique molecular index (to identify PCR errors following amplification). In some embodiments, the isolated DNA is ligated to nucleotide segments that facilitate downstream sequencing. In some embodiments, the isolated DNA is amplified using a two stage polymerase chain reaction (PCR) scheme, comprising: a) ligating a unique molecular index (UMI) to the DNA (to identify PCR errors), and b) and adding segments required for downstream sequencing. In some embodiments, amplified DNA is pooled. In some embodiments, the pooled DNA is sequenced using next generation sequencing.

[0097] In some embodiments, following the sorting step, an“enrichment ratio” (ER) is determined for each ABU by measuring the enrichment of an ABU. Such enrichment can be determined, for example, by NGS (next generation sequencing). In some embodiments, the ER is calculated according to the following formula: ER=log2(Fi/Fo) where Fs is the ABU frequency after i rounds of sorting and Fo is the frequency of the same ABU in the starting plurality of ABUs. Positive ERs indicate the mutation is enriched and negative ERs indicate the mutation is depleted.

[0098] In some embodiments, a light chain that is compatible with one or more different heavy chains is identified by assessing information stored in a Database, as described herein. In some embodiments, if the Database comprises data indicating: a) a first ABU is associated with a desired characteristic, wherein the first ABU binds a first epitope and comprises a variant or mutated light chain and a first heavy chain, and b) a second ABU is associated with a second desired characteristic, wherein the second ABU binds a second epitope and comprises a second heavy chain that is different from the first heavy chain, and wherein the second ABU comprises a variant or mutated light chain having the same amino acid sequence as the variant or mutated light chain of the first ABU; then the variant or mutated light chain is identified as being a light chain that is compatible with one or more different heavy chains.

[0099] In some embodiments, a variant or mutated light chain is identified as being a compatible light chain if: a) when paired with a first heavy chain to generate a first ABU, the first ABU binds a first epitope and is associated with a first desired characteristic, and b) when paired with a second heavy chain to generate a second ABU, the second ABU binds a second epitope and is associated with a second desired characteristic. In some embodiments, the first and second desired characteristics are a predetermined acceptable binding affinity for a first and second epitope, respectively. In some embodiments, the first and second characteristics are a predetermined acceptable ER for the first and second ABU. |Ό1QQ] In some embodiments, a variant or mutated light chain is identified as being compatible with a first for second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and the first (or second or further) heavy chain have at least a 10%, 20%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900% or 1000% higher frequency in a population of ABUs following a sorting process than a plurality of ABUs comprising a different variant or mutated light chain amino acid sequence and the first (or second or further) heavy chain in the same population of ABUs following the same sorting process. In some embodiments, the variant or mutated light chain is compatible with a first (or second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and a first (or second or further) heavy chain has a frequency in a population of ABUs following a sorting step that is not less than 200%, not less than 150%, not less than 100%, not less than 75%, not less than 50%, not less than 25%, or not less than 10% of the frequency of an ABU comprising a reference light chain amino acid sequence and the first (or second or further) heavy chain in a population of ABUs following the same sorting step. In some embodiments, the variant or mutated light chain is compatible with a first (or second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and a first (or second or further) heavy chain has at least a 10%, 20%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900% or 1000% greater enrichment ratio following a sorting step than an ABU comprising a reference light chain amino acid sequence and the first (or second or further) heavy chain following the same sorting step. In some embodiments, the variant or mutated light chain is compatible with a first (or second or further) heavy chain if a plurality of ABUs each comprising the variant or mutated light chain and a first (or second or further) heavy chain has an enrichment ratio following a sorting step that is not less than 200%, not less than 150%, not less than 100%, not less than 75%, not less than 50%, not less than 25%, or not less than 10% of the enrichment ratio of an ABU comprising a reference light chain amino acid sequence and the first (or second or further) heavy chain following the same sorting step. In some embodiments, a variant or mutated light chain is identified as being a compatible light chain if a first ABU comprising a first heavy chain and the variant or mutated light chain has an ER greater than 1, 1.5, 2, 2.5, 3, 3.5 or 4 for a first epitope, and a second ABU comprising a second heavy chain and the variant or mutated light chain has an ER greater than -1.5, -1.0, -0.5, 0, 0.5 or 1 for a second epitope. In some embodiments, a variant or mutated light chain is identified as being a compatible light chain if a first ABU comprising a first heavy chain and the variant or mutated light chain has an ER greater than or equal to 2 for a first epitope, and a second ABU comprising a second heavy chain and the variant or mutated light chain has an ER greater than or equal to -1.5 for a second epitope. In some embodiments, a variant or mutated light chain is not identified as being a compatible light chain if a first ABU comprising a first heavy chain and the variant or mutated light chain has an ER greater than 2, but a second ABU comprising a second heavy chain and the variant or mutated light chain has an ER less than -2.

[0101] It should be noted that, while the methods disclosed herein may refer to a“first” and “second” ABU, these methods can be expanded to generation and characterization of further ABUs (e.g., third, fourth, fifth, sixth, etc.).

Methods for Producing Multispecific ABMs

[0102] In some embodiments, the disclosure provides for a method for generating a multispecific ABM. In some embodiments, the multispecific ABM comprises two or more of any of the ABUs disclosed herein. In some embodiments, the multispecific ABM comprises two more ABUs, wherein each of the ABUs comprises a different heavy chain. In some

embodiments, each of the ABUs in the multispecific ABM comprises a compatible light chain, wherein each of the compatible light chains comprises the same amino acid sequence. In some embodiments, the compatible light chain was identified using any of the methods disclosed herein. In some embodiments, the compatible light chain is any of the variant or mutated light chains disclosed herein. In some embodiments, the compatible light chain was identified by assessing characteristic data associated with the compatible light chain that was stored in a Database. In some embodiments, the compatible light chain was identified using any of the screening methods disclosed herein.

[0103] In some embodiments, the disclosure provides for a method for generating a multi specific ABM comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain ammo acid sequences; wherein a variant or mutated light chain ammo acid sequence is identified as being compatible if: i) when paired with a first heavy chain ammo acid sequence to generate a first ABU, the first ABU binds a first epitope and is associated with a first desired characteristic; and ii) when paired with a second heavy chain amino acid sequence to generate a second ABU, the second ABU is capable of binding a second epitope and is associated with a second desired characteristic; and b) generating a multispecific ABM comprising the first ABU and the second ABU.

[0104] in some embodiments, the disclosure provides for a method for generating a multispecific ABM comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of mutated light chain amino acid sequences; wherein a mutated light chain amino acid sequence is identified as being compatible if: i) when paired with a first heavy chain ammo acid sequence to generate a first ABU, the first ABU is associated with a desired binding affinity to a first epitope; and ii) when paired with a second heavy chain ammo acid sequence to generate a second ABU, the second ABU is associated with a desired binding affinity to a second epitope; and b) generating a multispecific ABM comprising the first ABU and the second ABU.

[0105] In some embodiments, the disclosure provides for a method for generating a multispecific ABM comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of mutated light chain amino acid sequences; wherein a mutated light chain amino acid sequence is identified as being compatible if: i) when paired with a first heavy chain ammo acid sequence to generate a first ABU, the first ABU binds a first epitope and is associated with a first desired characteristic; and ii) when paired with a second heavy chain amino acid sequence to generate a second ABU, the second ABU binds a second epitope and is associated with a second desired characteristic; and b) generating a multispecific ABM comprising the first ABU and the second ABU; wherein the complementari ty determining regions (CDRs) of the multispecific ABU are not further optimized for the desired characteristic.

[0106] In some embodiments, the disclosure provides for a method for generating a multispecific ABM comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of mutated light chain ammo acid sequences; wherein the mutated light chain ammo acid sequences in the Database were mutated as compared to a reference light chain amino acid sequence; wherein a mutated light chain ammo acid sequence is identified as being compatible if: i) when paired with a first heavy chain amino acid sequence to generate a first ABU, the first ABU is capable of binding a first epitope and is associated with a first desired characteristic; and ii) when paired with a second heavy chain ammo acid sequence to generate a second ABU, the second ABU is capable of binding a second epitope and is associated with a second desired characteristic; and b) generating a multispecific ABM comprising the first ABU and the second ABU.

[0107] In some embodiments, the disclosure provides for a method for generating a multispecific ABM comprising a first ABU having a first desired characteristic and a second ABU having a second desired characteristic, the method comprising: a) screening a library of first ABUs for a first desired characteristic, wherein the library comprises a plurality of first ABUs, wherein each of the first ABUs comprises: i) the same first heavy chain (e.g., heavy chain variable region) amino acid sequence and ii) a unique variant light chain (e.g., light chain variable region) ammo acid sequence relative to a reference light chain ammo acid sequence; and b) generating a multispecific ABM comprising: 1) a first ABU identified from step (a) having the desired characteristic; and 2) a second ABU, wherein the second ABU has a second desired characteristic and comprises: (x) a second heavy chain (e.g., heavy chain variable region) ammo acid sequence and (y) the same variant light chain (e.g., light chain variable region) acid sequence as the first ABU.

[0108] In some embodiments, any of the variant or mutated light chains, reference light chain ammo acid sequences, heavy chains, ABUs, ABMs, Databases, and/or methods disclosed herein may be used in any of the methods for generating any of the multispecific ABMs disclosed herein.

[0109] In some embodiments, the binding affinity of any of the ABUs or ABMs disclosed herein for a particular epitope or epitopes can be confirmed or further screened using any immunological or biochemical based method known in the art. For example, specific binding of an ABM or ABU for a particular epitope or epitopes may be determined, for example, using immunological or biochemical based methods such as, but not limited to, an ELISA assay, SPR assays, immunoprecipitation assay, affinity chromatography, and equilibrium dialysis as described above. Immunoassays that can be used to analyze immunospecific binding and cross- reactivity of the ABU s or ABMs include, but are not limited to, competitive and non-competiti ve assay systems using techniques such as Western blots, RLA, ELISA (enzyme linked

immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays.

[0110] In some embodiments, any of the ABMs or ABUs disclosed herein comprises an antibody. In some embodiments, the antibody can be modified to generate an antigen- binding fragment, as described herein, and/or manipulated using known techniques in the art to generate a multispecific ABM as described herein. For example, cross-linking methods can be used to generate a multispecific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl reactive group as described in, e.g., U.S. Pat. No. 4,433,059; multispecific antibody determinants can be generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycles of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., three Fab' fragments can be cross-linked through sulfhdryl reactive groups, as described in, e.g., U.S. Pat. No. 5,273,743.

[0111] The multispecific ABMs disclosed herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry . For example, a nucleic acid encoding the multispecific ABM (as a single multifunctional polypeptide, or as separate molecules of a multimeric complex - e.g., one ABU separately from the other ABU) can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system, such that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

[0112] Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian ceils. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) MolAppl Genet 1 :327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements that confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal vir uses, such as bov ine papillomavirus (Sarver et al. (1982) Proc Nail Acad Sei USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81 : 1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0113] The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPCti precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0114] Appropriate host ceils for the expression of ABUs and ABMs include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. con, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect ceils such as SF9, mammalian ceil lines (e.g., human ceil lines), as well as primary cell lines.

[0115] The ABMs or ABUs can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the antibodies or fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression vary with the choice of the expression vector and the host cell and are easily ascertained by one skilled in the art through routine

experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g. , Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are known in the art (see Ausubei et al. (1988) Current Protocols in Molecular Biology, Wiley & Sons; and Green and Sambrook (2012) Molecular Cloning— A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells vary depending on a number of factors, and may be easily optimized as needed. An ABM or ABU can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220). [0116] Following expression, the ABU or ABM can be isolated. An ABU or ABM can be isolated or purified in a variety of ways known in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein- A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) Protein Purification, 3^rd edition, Springer- Ver lag, New York City, New York.

The degree of purification necessary varies depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof is necessary-.

[0117] Methods for determining the yield or purity of a purified antibody or fragment thereof are known m the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).

[0118] In some embodiments, any of the multispecific ABMs disclosed herein can be modified as a single multifunctional polypeptide or as separate molecules of a multimeric complex - e.g., one ABU separately from the other ABU. The modifications can be covalent or non-covalent modifications. Such modifications can be introduced into the ABUs or ABMs by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or fragments.

[0119] In some embodiments, the ABUs or ABMs can be conjugated to a heterologous moiety. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG (DYKDDDDK) (SEQ ID NO: 20), poly histidme (6-His; HHHHHH (SEQ ID NO: 21)), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 22)), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the ABUs or ABMs. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). Suitable radioactive labels include, e.g, ⁱ²P, ³³P, ⁱ⁴C,

and -Ή. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488,

phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of di ethylene triamine pentaacetic acid (DTP A) or tetraazacyclododecane- 1,4, 7,10- tetraacetic acid (DOTA). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0120] Two proteins (e.g., an ABU or ABM and a heterologous moiety') can be cross-linked using any of a number of known chemical cross linkers. Examples of such cross linkers are those that link tw^ro ammo acid residues via a linkage that includes a“hindered” disulfide bond.

In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl- a-methyl-a(2-pyndyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other.

Heterobifunctioiial reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which link two ammo groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4- bis-maleimidobutane), an ammo group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p- azidosalicylamidojbutylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl g!yoxal monohydrate).

[0121] In some embodiments, a radioactive label can be directly conjugated to the amino acid backbone of the ABU or ABM. Alternatively, the radioactive label can be included as part of a larger molecule (e.g., ^l25I in meta-[^!25I]iodophenyl~N-hydrox>'Suceinimide ([^{1 5}I]mIPNHS), which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al. (1997) JNucl Med 38:1221-1229) or chelate (e.g, to DOTA or DTP A), which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels or larger molecules/chelates containing them to the ABUs or ABMs described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and/or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U.S. Patent No. 6,001,329).

[0122] Methods for conjugating a fluorescent label (sometimes referred to as a fluorophore) to a protein (e.g., an ABU or ABM) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some embodiments, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC. Suitable conjugation methods involve incubating an ABU or ABM with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) Handbook of

Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons.

[0123] In some embodiments, the ABUs or ABMs can be modified, e.g., with a moiety that improves the stabilization and/or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues. For example, the ABU or ABM can be PEGylated as described in, e.g., Lee et al. (1999 ) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476, or HESylated (Fresenius Kabi, Germany) (see, e.g., Pavisic et al. (2010) IntJPharm 387(1-2): 110- 119). The stabilization moiety can improve the stability, or retention of, the ABU or ABM by at least 1.5 (e.g., at least 2, 5, 10, 1 5, 20, 25, 30, 40, or 50 or more) fold.

[0124] In some embodiments, the ABUs or ABMs thereof described herein can be glycosylated. In some embodiments, an ABU or ABM described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the ABU or ABM has reduced or absent giycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., U.S. Patent No. 6,933,368; Wright et al. (1991) EMBO J 10(10): 2717-2723; and Co et al. (1993) Mol Immunol 30: 1361.

V. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[0125] Compositions comprising any of the multispecific ABMs of the present disclosure and a pharmaceutically acceptable carrier are also provided. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and/or adjuvant to be used with the methods disclosed herein. Such compositions can be used in a subject having condition that would benefit from the multispecific ABMs described herein.

[0126 j In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous (s.c.) and/or intravenous (I.V.) administration. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the

composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl- beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorhitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); deliver vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Allen (2012) Remington The Science and Practice of Pharmacy, 22d Edition,

Lloyd V, Allen, ed., The Pharmaceutical Press). In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, deliver} format and desired dosage. See, for example, Allen (2012) Remington The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and/or rate of in vivo clearance of the multispecific ABM.

[0127] In certain embodiments, the primary vehicle or carrier in a pharmaceutical

composition can be either aqueous or non-aqueous m nature. For example, in certain

embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary- vehicles. In certain embodiments,

pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In certain embodiments, a composition comprising any of the multispecific ABMs disclosed herein can be prepared for storage by mixing the selected composition ha ving the desired degree of purity with optional formulation agents (see Allen (2012) Remington The Science and Practice of

Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising any of the multispecific ABMs disclosed herein can be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0128] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

[0129] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

[0130] In certain embodiments when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a multispecific ABM, in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which a multispecific ABM is formulated as a sterile, isotonic solution, and properly preserved. In certain

embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as poly lactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection in certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0131] In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, a multispecific ABM can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising a multispecific ABM can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in PCX Application No.

PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

[0132] In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, a multispecific ABM that is administered in this fashion can be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemie degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of a multispecific ABM. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0133] In certain embodiments, a pharmaceutical composition can involve an effective quantity of a multispecific ABM in a mixture with non-toxic excipients suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water or other appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc. [0134] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving a multispecific ABM in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCX

Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeabie polymer matrices in the form of shaped articles, e.g., films, or microcapsuies. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and European Patent No. EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1993) Biopolymers 22: 547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al. (1981) J Biomed Mater Res 15: 167-277; and Langer (1982) ( ^'hem Tech 12:98-105), ethylene vinyl acetate (Langer et al.) or poly-D(-)-3-hydroxybutyric acid (European Patent No, EP 133,988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. (See, e.g., Eppstein et al. (1985) Proc. Natl. Acad. Set. USA 82:3688-3692; European Patent Nos. EP 036,676; EP 088,046; and EP 143,949.

[0135] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceabie by a hypodermic injection needle.

[0136] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready- to-use form or in a form (e.g , lyophilized) that is reconstituted prior to administration.

[0137] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.

[0138] In certain embodiments, the effective amount of a pharmaceutical composition comprising a multispecific ABM to be employed therapeutically depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which a multispecific ABM is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0139] The clinician also selects the frequency of dosing, taking into account the

pharmacokinetic parameters of the multispecific ABM in the formulation used. In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0140] In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, mtraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of the combination therapy may be administered by different routes.

[0141] In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0142] In certain embodiments, it can be desirable to use a pharmaceutical composition comprising a multispecific ABM in an ex vivo manner. In such instances, ceils, tissues (including, e.g., blood) and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising a multispecific ABM after which the cells, tissues and/or organs are subsequently implanted back into the patient.

[0143] In certain embodiments, a multispecific ABM can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides. In certain embodiments, such ceils can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the ceils can be immortalized. In certain embodiments, in order to decrease the chance of an immunological response, the ceils can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi- permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

VL EQUIVALENTS AND SCOPE

[0144] Those skilled in the art. will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0145] In the claims, articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. [0146] It is also noted that the term“comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term“comprising” is used herein, the term“consisting of’ is thus also encompassed and disclosed.

[0147] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0148] In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary' skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions (e.g., any antibiotic, therapeutic or active ingredient: any method of production: any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0149] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit in its broader aspects.

[0150] While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with reference to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope.

[0151] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. The following description provides further non-limiting examples of the disclosed compositions and methods.

VIL EXAMPLES

[0152] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments of the present invention, and are not intended to limit the invention. Example 1 ; Identification of affinity-matured cLC via analysis of DMS data

[0153] A simplified outline of an approach to generating a multispecific ABM composing a common light chain (cLC) is illustrated in Figure 1.

[0154] One approach to generating affinity-matured cLCs is to construct cLCs with mutations identified through analysis of paired DMS data. In this example, cLC DMS data was generated from phage panning of anti-A and anti-B libraries (Figure 2). This data is presented as enrichments ratios for each possible single amino acid substitution in the cLC CDRs. Enrichment ratios (ERs) were calculated by log? transformation of the ratio of the frequency of a mutation after panning to the frequency of that same mutation m the starting library. Positive ERs indicated the mutation is enriched and negative ERs indicate the mutation is depleted. By careful examination and comparison of the ER heatmaps for anti-A and anti-B cLC DMS several mutations were further examined (Figures 2A and 2B). In this study, compatible affinity- improving mutations (Figure 2B, examples shown as grey-filled, thick-outlined symbols) w¾re defined as those that have an ER>2 for one target and an ER>~ 1.5 for the other. On the other hand, non-compatible mutations (Figure 2B, black-filled symbols) were defined as those with ER>2 for one target and ER<~2 for the other. Novel cLCs were built to incorporate one or more of the compatible mutations and paired with either the wild type (WT) anti-A and anti-B variable heavy chain domains (VHs) or the Affinity matured (AffMat) anti-A and anti-B VHs. These pairings were produced as monoclonal IgG and binding kinetics were assayed via surface plasmon resonance (SPR). Equilibrium dissociation constant (KD) data for selected pairings is presented in Figure 2C.

Example 2: Leveraging DMS for focused cLC combinatorial library design

[0155] While Example 1 was successful at generating affinity matured cLC leads, additional mutations and potential combinations thereof were not tested in that example. An alternative approach is to leverage the DMS data to construct a focused cLC combinatorial library and use subsequent mammalian display sorting in a bispecific format to identify optimal cLCs. In tins example, cLC DMS data was generated from phage panning of an anti-C library (Figure 3 A) and mammalian display sorting of an anti-D library (Figure 3B and C, see methods for sorting details). These data were used to identify a list of compatible mutations at each CDR position and generate a focused combinatorial library as described in the methods section. Methods

Library Construction

[0156] cLC DMS Libraries: CDR single site saturation libraries (SSL) for Vk! .39 and Vk3.15 cLCs (SEQ ID NOs: 1 and 2, respectively) were constructed via standard molecular cloning procedures as follows. A series of oligonucleotides introducing single NNK degenerate codons (N=G, A, T or C; K G or T) at each position within CDRL1, L2, or L3 (IDT) were pooled by CDR and stitched together with wild-type framework and CDR DNA via 3 separate overlap- extension PCRs to generate full-length variable regions. Reaction products of the correct size (~450bp) were isolated via gel electrophoresis and CDRLl, CDRL2, CDRL3 mutated fragments combined. This final product may serve as the stock cLC DMS library and can be paired with any desired VH via Gibson cloning into the display plasmid of choice ( e.g . phage or mammalian display). Phage libraries express monovalent Fabs fused to the pill coat protein. Mammalian display libraries express full-length mAbs on the HEK cell surface.

[0157] Combinatorial libraries: Deep mutational scanning (DMS) data w¾s used to generate combinatorial library designs used in Example 2. Anti-C ERs from phage panning (Figure 3 A) were compared to anti-D ERs generated from mammalian display of both high and medium affinity sorts (Figure 3B and C). ER cutoffs were applied to generate a list of compatible mutations: (1) Anti-C ER>2 and an Anti-D High affinity ER>-1 , (2) Anti-C ER>2 and an Anti-D medium affinity ER>-1.5, (3) Anti-D High affinity ER>2 and an Anti-C ER>-1.5, (4) Anti-D medium affinity ER>2 and an Anti-C ER>~1.5. This cutoff analysis resulted in a list of desired amino acid diversity at each position within the CDRs. Next, degenerate codons were identified which captured as much of the desired diversity at each position while minimizing off-target mutations and keeping the theoretical library diversity within the mammalian display

transformation efficiency (upper limit of about 10⁶- 1 O '). Oligonucleotides with the selected degenerate codons were obtained from IDT and stitched together with wildtype DNA as described above.

[0158] Next Generation Sequencing (NGS) sample preparation and data processing:

Antibody sequences were isolated from the phage or mammalian plasmid DNA of the starting library and each panning or sorting round. These DNA isolates were then amplified using two stage PCR schema where the first stage ligated a unique molecular index (UMI) and the second stage added segments required for downstream sequencing. Amplified DNA libraries were then quality checked and pooled before sequencing on a single MiSeq flowcell in a 2x300 cycle paired-end run.

[0159] The MiSeq run generated approximately 12M sequences (~1 5M per sample). The paired sequences were quality filtered, joined, and aligned to the reference antibody sequence. Each sample in the run was demultiplexed using the unique sequence encoded during the first stage PCR. Individual sequences were then processed using the UMI and mutation patterns to remove errors introduced from amplification to sequencing. Aggregate single ammo acid mutation frequencies within the 3 CDRs were used to calculate enrichment ratio (ER) according to the following formula: ER=log2(Fi/Fo) where Fi is the mutation frequency after i rounds of panning or sorting and Fo is the frequency of the same mutation in the starting library.

[0160] Cloning and expression of compatible cLCs: Compatible mutations are identified through analysis of paired DMS data as described in Example 1. Light chain (LC) DNA fragments incorporating one or more compatible mutations were obtained from IDT and Gibson cloned into a mammalian expression plasmid. The LC plasmid can then be combined with the desired monoclonal or bi-/multi-specific HCs and transiently transfected into HEK cells. The resultant expression products are purified via standard techniques ( e.g . Protein A

chromatography) and can be used for desired affinity' measurements and activity assays.

[0161] Phage Panning of cLC DMS libraries: Phage panning was used to enrich the library' for mutations with improved affinity'. The library was transformed into TGI e coli and Ml 3 helper-phage infection used to generate phage displaying Fabs fused to the pill coat protein. Three to four rounds of panning were routinely carried out with increased stringency in each round by reducing antigen concentration and incorporating off-rate competition steps. A typical panning round involved the following steps: (1) Precipitation of library phage from overnight culture of helper-phage infected e. coli, (2) Incubation of phage with antigen-coated magnetic beads, (3) Washing, (4) Off-rate competition, (5) Elution, (6) library recovery via infection of e. coli.

[0162] VH optimization alone yielded significant improvements in affinity (88-fold for anti-A and 16-fold for anti-B). The affinity data for pairing of these AffMat VHs with DMS-guided compatible cLCs indicated significant additional affinity improvements (up to 5-fold) against both antigens which behave generally as predicted. For example, mutations predicted to be incompatible for anti-B (S91G, Y49K) significantly improved anti-A binding while completely knocking out anti-B binding. On the other hand, mutations which were predicted to be compatible based on the DMS data analysis (Figure 2A and B) were found to improve the affinity for both anti-A and anti-B binding beyond that achieved with VH mutations alone.

Affinity measurements

[0163] The resulting antibodies from different selection rounds were plotted on kd/'ka double log plots. Apparent association and dissociation kinetic rate constants (ka and kd values) were determined on an SPRi reader (MX96, Carterra, Salt Lake City, UT)) in a running buffer of PBS- C 0.01%. Antibodies were covalently printed on a Carboxymethyldextran hydrogel 50L chip (XanTec bioanalytics GmbH, Dusseldorf, Germany) on a CFM (Carterra). Freshly mixed activating reagents (150 ml 0.4 M EDC and 150 ml 0.1 M sulfo-NHS in H20) were used to activate the surface of the SPR substrate for 7 minutes. Antibodies at 10 mg/ml, m acetic acid buffer pH 4.5, were used for printing for 15 minutes. The printed chip was then quenched on an SPRi reader (MX96, Carterra) with 1 M ethanolamine for 15 minutes. For kinetics analysis, purified recombinant His tagged human CD! 37 (0, 2.05, 5.12, 12.8, 32, 80, 200, 500 nM) w¾s injected sequentially. For each concentration, there was 5 minutes of association followed by 10 minutes of dissociation. Data were processed and analyzed in SPR Inspection Tool and Scrubber software (Biosensor Tools LLC, Salt Lake City, UT). The kinetic data were referenced with the interstitial reference spots and double-referenced to a buffer cycle, and then fit globally to a 1 : 1 binding model to determine their apparent association and dissociation kinetic rate constants (ka and kd values). The ratio kd/ka was used to derive the KD value of each antigen/m Ab interaction, i.e. KD^::=kd/ka.

Mammalian display sorting of cLC libraries

[0164] Monoclonal display: As an alternative to phage display, mammalian display was also used to sort for clones with desired affinity. For generation of initial DMS data the cLC DMS antibody library was transfected into an acceptor cell line that displays the full antibody on the surface of HEK cells. Cells were incubated with antigen and sorted with 3 different gates: high, medium, and low, representing populations that showed higher, same, or lower binding than WT. Cells were expanded and sorted again using the same staining condition and gates for one additional round.

Claims

1. A method for generating a multispecific antigen-binding molecule (ABM) comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain am o acid sequences; wherein the variant or mutated light chain amino acid sequence is identified as being compatible if:

i) vtiien paired with a first heavy chain ammo acid sequence to generate a first antigen-binding unit (ABU), the first ABU binds a first epitope and is associated with a first desired characteristic; and

ii) when paired with a second heavy chain amino acid sequence to generate a second ABU, the second ABU binds a second epitope and is associated with a second desired characteristic; and

b) generating a multispecific ABM comprising the first ABU and the second ABU.

2. A method for generating a multispecific antigen-binding molecule (ABM) comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain ammo acid sequences; wherein a variant or mutated light chain amino acid sequence is identified as being compatible if:

i) when paired with a first heavy chain ammo acid sequence to generate a first antigen-binding unit (ABU), the first ABU is associated with a desired binding affinity to a first epitope; and

ii) when paired with a second heavy chain amino acid sequence to generate a second ABU, the second ABU is associated with a desired binding affinity to a second epitope; and

b) generating a multispecific ABM comprising the first ABU and the second ABU.

3. A method for generating a multispecific antigen-binding molecule (ABM) comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain ammo acid sequences; wherein a variant or mutated light chain ammo acid sequence is identified as being compatible if:

i) when paired with a first heavy chain ammo acid sequence to generate a first antigen-binding unit (ABU), the first ABU binds a first epitope and is associated with a first desired characteristic; and

b) generating a multispecific ABM comprising the first ABU and the second ABU;

wherein the complementarity determining regions (CDRs) of the multispecific ABM are not further optimized for the desired characteristic.

4. A method for generating a multispecific antigen-binding molecule (ABM) comprising a common light chain that is compatible with two or more different heavy chains, comprising: a) identifying a compatible light chain amino acid sequence from a Database of variant or mutated light chain ammo acid sequences; wherein the variant or mutated light chain amino acid sequences in the Database were mutated as compared to a reference light chain amino acid sequence; wherein a variant or mutated light chain amino acid sequence is identified as being compatible if:

b) generating a multispecific ABM comprising the first ABU and the second ABU.

5. The method of any one of claims 1-3, wherein the variant or mutated light chain amino acid sequences in the Database were mutated as compared to a reference light chain amino acid sequence.

6. The method of claim 4 or 5, wherein the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

7. The method of claim 4 or 5, wherein the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2.

8. The method of claim 4 or 5, wherein the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3.

9. The method of claim 4 or 5, wherein the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4.

10. The method of claim 4 or 5, wherein the Database comprises a plurality of variant or mutated light chain amino acid sequences, wherein each of the variant or mutated light chain amino acid sequences in the plurality of sequences comprises at least one amino acid alteration as compared to a CDR1 , CDR2 and/or CDR3 of a reference light chain amino acid sequence.

1 1. The method of any one of claims 1 -9, wherein the Database comprises a plurality of variant or mutated light chain ammo acid sequences, wherein each of the variant or mutated light chain amino acid sequences in the plurality of sequences comprises at least one ammo acid alteration as compared to the ammo acid sequences of any one of SEQ ID NOs: 5-19.

12. The method of any of claims 1-11, wherein the Database composes a plurality of variant or mutated light chain ammo acid sequences, wherein each of the variant or mutated light cham amino acid sequences in the plurality of sequences comprises at least one ammo acid alteration in the framework region as compared to the framework region of a reference light cham amino acid sequence.

13. The method of any one of claims 4-12, wherein the variant or mutated light cham amino acid sequences in the Database were mutated as compared to a single reference light chain ammo acid sequence.

14. The method of any one of claims 1-13, wherein one or more of the variant or mutated light cham amino acid sequences in the Database were determined using next generation sequencing.

15. The method of any one of claims 1 or 3-14, wherein the first and/or second desired characteristic is desired binding affinity'.

16. The method of any one of claims 1-15, wherein the variant or mutated light chain amino acid sequences were generated using single-site saturation.

17. The method of any one of claims 1-16, wherein the variant or mutated light chain amino acid sequences were characterized using deep mutational scanning.

18. The method of any one of claims 1 -17, wherein the Database comprises variant or mutated light chain ammo acid sequences consisting of 1 -5 mutated ammo acid residues as compared to a reference light chain amino acid sequence.

19. The method of any one of claims 1 -18, wherein the Database comprises variant or mutated light cham amino acid sequences consisting of one mutated amino acid residue as compared to a reference light chain amino acid sequence.

20. The method of any one of claims 1 -19, wherein each of the variant or mutated light chain amino acid sequences in the Database differs from the other variant or mutated light chain ammo acid sequences in the Database by no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amnio acids.

21. The method of any one of claims 1-20, wherein the Database comprises at least 50 different variant or mutated light chains amino acid sequences.

22. The method of any one of claims 1-20, wherein the Database comprises at least 100 different variant or mutated light chains amino acid sequences.

23. The method of any one of claims 1-20, wherein the Database comprises at least 200 different variant or mutated light chains amino acid sequences.

24. The method of any one of claims 1-20, wherein the Database comprises at least 500 different variant or mutated light chains amino acid sequences.

25. The method of any one of claims 1 and 3-24, wherein the first desired characteristic is a desired binding affinity of the first ABU for a first epitope.

26. The method of any one of claims 1 and 3-25, wherein the second desired

characteristic is a desired binding affini ty of the second ABU for a second epitope.

27. The method of claim 25 or 26, wherein the desired binding affinity of the first ABU for the first epitope is measured m terms of an enrichment ratio (ER).

28. The method of claim 26 or 27, wherein the desired binding affinity of the second ABU for the second epitope is measured in terms of an enrichment ratio (ER).

29. The method of any one of claims 2 and 26-28, wherein the desired binding affinity of the first ABU for the first epitope is measured in terms of an enrichment ratio (ER), and wherein the desired binding affinity of the second ABU for the second epitope is measured in terms of an enrichment ratio.

30. The method of claim 29, wherein the variant or mutated light chain is identified as being a compatible light chain if the first ABU has an ER greater than 1, 1.5, 2, 2.5, 3, 3.5 or 4 for the first epitope, and the second ABU has an ER greater than -1.5, -1.0, -0.5, 0, 0.5 or 1 for the second epitope.

31. The method of claim 29, wherein the variant or mutated light chain is identified as being a compatible light chain if the first ABU has an ER greater than 2 for the first epitope, and the second ABU has an ER greater than -1.5 for the second epitope.

32. The method of claim 29, wherein the variant or mutated light chain is identified as being a compatible light chain if the first ABU has an ER greater than 2 for the first epitope, and the second ABU has an ER greater than -2 for the second epitope.

33. The method of any one of claims 1-32, wherein the first heavy chain was affinity matured for binding to the first epitope.

34. The method of any one of claims 1 -33, wherein the second heavy chain was affinity matured for binding to the second epitope.

35. A method for generating a multispecific antigen-binding molecule (ABM) comprising a first antigen-binding unit (ABU) having a first desired characteristic and a second ABU having a second desired characteristic, the method comprising:

a) screening a library of first ABUs for a first desired characteristic, wherein the library comprises a plurality of first ABUs, wherein each of the first ABUs comprises:

i) the same first heavy chain amino acid sequence and ii) a unique variant or mutated light chain ammo acid sequence relative to a reference light chain ammo acid sequence; and

b) generating a multispecific ABM comprising:

1 ) a first ABU identified from step (a) having the desired characteristic; and

2) a second ABU, wherein the second ABU has a second desired characteristic and comprises:

(x) a second heavy chain amino acid sequence and

(y) the same variant light chain amino acid sequence as the first ABU.

36. The method of claim 35, wherein each variant or mutated light chain amino acid sequence comprises one or more amino acid substitutions, deletions, or insertions relative to a reference light chain ammo acid sequence.

37. The method of claim 36, wherein each variant or mutated light chain amino acid sequence comprises fewer than 20, fewer than 15, fewer than 10, fewer than 5; no more than five, 10, 15, or 20 amino acid substitutions, deletions, or insertions relative to a reference light chain amino acid sequence.

38. The method of any one of claims 35-37, wherein the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 1

39. The method of any one of claims 35-37, wherein the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2.

40. The method of any one of claims 35-37, wherein the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 3.

41. The method of any one of claims 35-37, wherein the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4.

42. The method of any one of claims 35-37, wherein a plurality of the variant or mutated light chain amino acid sequences in the library comprise at least one ammo acid alteration as compared to a CDRl, CDR2 and/or CDR3 of a reference light chain amino acid sequence.

43. The method of any one of claims 35-42, wherein a plurality of the variant or mutated light chain amino acid sequences in the library comprise at least one ammo acid alteration as compared to the framework region of a reference light chain amino acid sequence.

44. The method of any one of claims 35-37, wherein a plurality of the variant or mutated light chain amino acid sequences in the library comprises at least one amino acid alteration as compared to the ammo acid sequences of any one of SEQ ID NOs: 5-19

45. The method of any one of claims 35-44, wherein the variant or mutated light chain amino acid sequences in the library were mutated as compared to a single reference light chain amino acid sequence.

46. The method of any one of claims 35-45, wherein the variant or mutated light chain amino acid sequences were generated using single-site saturation.

47. The method of any one of claims 35-46, wherein the variant or mutated light chain amino acid sequences were characterized using deep mutational scanning.

48. The method of any one of claims 35-47, wherein the screening step a) comprises phage display, yeast display, and/or mammalian display.

49. The method of any one of claims 35-48, wherein the screening step of a) comprises assaying the binding affinity of the first ABUs for a first antigen.

50. The method of claim 49, wherein the binding affinity of the first ABUs is compared to the binding affinity of a reference antibody or antigen-binding fragment for the same antigen.

51. The method of any one of claims 35-50, wherein the screening step a) comprises panning and/or sorting the first ABUs based on whether the first ABUs are determined to have the desired characteristic.

52. The method of any one of claims 35-51, wherein the method further comprises determining the sequence of at least a portion of a first ABU determined to have the first desired characteristic, or determining the nucleotide sequence of at least a portion of the nucleic acid encoding the first ABU determined to have the first desired characteristic.

53. The method of claim 52, wherein next generation sequencing is used to determine the nucleotide sequence of at least a portion of the nucleic acid encoding the first ABU.

54. The method of any one of claims 35-53, wherein the first desired characteristic is a desired binding affinity for a first epitope, and wherein the second desired characteristic is a desired binding affinity for a second epitope.

55. The method of claim 54, wherein the desired binding affinity of the first ABU for the first epitope is measured in terms of an enrichment ratio (ER), and wherein the desired binding affinity of the second ABU for the second epitope is measured in terms of an enrichment ratio.

56. The method of claim 55, wherein the first ABU is determined to have the desired characteristic if the first ABU has an ER greater than 1, 1.5, 2, 2.5, 3, 3.5 or 4 for the first epitope, and the second ABU is determined to have the desired characteristic if the second ABU has an ER greater than -1.5, -1.0, -0.5, 0, 0.5 or 1 for the second epitope.

57. The method of claim 55, wherein the first ABU is determined to have the desired characteristic if the first ABU has an ER greater than 2 for the first epitope, and the second ABU is determined to have the desired characteristic if the second ABU has an ER greater than -1.5 for the second epitope.

58. The method of claim 55, wherein the first ABU is determined to have the desired characteristic if the first ABU has an ER greater than 2 for the first epitope, and the second ABU is determined to have the desired characteristic if the second ABU has an ER greater than -2 for the second epitope.

59. The method of any one of claims 35-58, wherein the first heavy chain was affinity matured for binding to the first epitope.

60. The method of any one of claims 35-59, wherein the second heavy chain was affinity matured for binding to the second epitope.

61. The methods of any one of claims 35-60, wherein data obtained from the screening step a) are stored in a Database.

62. The method of any one of claims 1-61, wherein the method further comprises generating an expression vector comprising one or more nucleotide sequences encoding the multispecific ABM.

63. The method of claim 62, wherein the method further comprises expressing the ABM from a cell.

64. A multispecific ABM generated using any of the methods of claims 1-63.

65. The multispecific ABM of claim 64, wherein the multispecific ABM is a bispecific

ABM.

66. The multispecific ABM of claim 64, wherein the multispecific ABM is a irispecific

ABM.

67. The multispecific ABM of any one of claims 64-66, wherein the ABM is human or humanized.

68. The multispecific ABM of any one of claims 64-66, wherein the ABM is an antibody.

69. A nucleic acid or plurality of nucleic acids encoding the ABM of any one of claims

64-68.

70. A vector comprising the nucleic acid or plurality of nucleic acids of claim 69.

71. A cell comprising the nucleic acid or plurality of nucleic acids of claim 69 or the vector of claim 70.

72. A method for creating a Database of variant or mutated light chains, comprising: a) providing a library comprising a plurality of different variant or mutated light chains; wherein each variant or mutated light chain in the library comprises at least one amino acid alteration as compared to a reference light chain ammo acid sequence;

b) providing a set of heavy chains, wherein each of the heavy chains comprises the same amino acid sequence;

c) separately pairing each of the different variant or mutated light chains with heavy chains from the set of heavy chains, thereby generating a library of antigen-binding units (ABUs) that bind an antigen;

d) screening the ABUs from the library of ABUs for a desired characteristic,

e) optionally repeating steps c) and d) with a different set of heavy chains each having an identical amino acid sequence; f) storing in a Database the data obtained from the screening step d) and optionally from the screening step m e).

73. The method of claim 72, wherein each variant light chain amino acid sequence comprises one or more ammo acid substitutions, deletions, or insertions relative to a reference light chain ammo acid sequence.

74. The method of claim 72, wherein each variant light chain amino acid sequence comprises fewer than 20, fewer than 15, fewer than 10, fewer than 5; no more than five, 10, 15, or 20 amino acid substitutions, deletions, or insertions relative to a reference light chain ammo acid sequence.

75. The method of any one of claims 72-74, wherein the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

76. The method of any one of claims 72-74, wherein the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2.

77. The method of any one of claims 72-74, wherein the reference light chain amino acid sequence comprises an ammo acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ

ID NO: 3

78. The method of any one of claims 72-74, wherein the reference light chain amino acid sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 4.

79. The method of any one of claims 72-74, wherein a plurality of the variant or mutated light chain amino acid sequences in the librar comprise at least one amino acid alteration as compared to a CDRl, CDR2 and/or CDR3 of a reference light chain amino acid sequence.

80. The method of any one of claims 72-74, wherein a plurality of the variant or mutated light chain amino acid sequences in the librar comprise at least one amino acid alteration as compared to the framework regions of a reference light chain amino acid sequence.

81. The method of any one of claims 72-74, wherein a plurality of the variant or mutated light chain amino acid sequences in the library comprises at least one amino acid alteration as compared to the ammo acid sequences of any one of SEQ ID NOs: 5-19.

82. The method of any one of claims 72-81, wherein the variant or mutated light chain amino acid sequences in the library' were mutated as compared to a single reference light chain ammo acid sequence.

83. The method of any one of claims 72-82, wherein the variant or mutated light chain amino acid sequences were generated using single-site saturation.

84. The method of any one of claims 72-83, wherein the variant or mutated light chain ammo acid sequences were characterized using deep mutational scanning.

85. The method of any one of claims 72-84, wherein the screening step d) and/or e) comprises phage display, yeast display, and/or mammalian display.

86. The method of any one of claims 72-85, wherein the screening step of d) comprises assaying the binding affinity' of the ABUs for a first antigen.

87. The method of claim 86, wherein the binding affinity of the ABUs in step d) are compared to the binding affinity of a reference ABU for the same antigen.

88. The method of any one of claims 72-87, wherein the screening step d) comprises panning and/or sorting the ABUs based on whether the ABUs are determined to have the desired characteristic.

89. The method of any one of claims 72-88, wherein the method further comprising step e), wherein step e) comprises repeating steps c) and d) with a different set of heavy chains each having an identical amino acid sequence.

90. The method of any one of claims 72-89, wherein the heavy chains of step b) were affinity^ matured for binding to an epitope.

91. The method of any one of claims 72-90, wherein the heavy chains of step e) were affinity matured for binding to an epitope.

92. A database comprising:

a) a plurality of variant or mutated light chain amino acid sequences;

b) a plurality' of heavy chain amino acid sequences;

c) binding affinity data of a plurality' of antigen-binding unit (ABUs) for one or more antigens, wherein each of the ABUs comprises a variant or mutated light chain amino acid sequence selected from a) and a heavy chain ammo acid sequence selected from b).