AU2023202781B2 - Common light chain mouse - Google Patents

Abstract

A genetically modified mouse is provided, wherein the mouse is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa (K) constant gene at the endogenous mouse K locus, wherein the mouse expresses a reverse chimeric antibody having a light chain variable domain derived from one of only two human light chain variable region gene segments and a mouse K constant domain, and a human heavy chain variable domain and a mouse heavy chain constant domain, from an endogenous mouse heavy chain locus. Bispecific epitope-binding proteins that are fully human are provided, comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments.

Description

COMMONLIGHT COMMON CHAIN MOUSE LIGHTCHAIN MOUSE

Thepresent

[0001] The

[0001] presentapplication divisional application applicationisisa adivisional application from from Australian patentapplication Australian patent application number 2020260398, number 2020260398,which which is turn is in in turn a divisionalapplication a divisional application from from Australian Australian patent patent application number2018201014, application number 2018201014, whichwhich is in is in aturn turn a divisional divisional application application from Australian from Australian

patent application patent application number number 2016203148, 2016203148, which which in turn in is turn is a divisional a divisional application application from from Australian patent Australian patent application application number number2014200245, 2014200245, whichwhich in turn in turn is a is a divisional divisional application application

from Australian from Australian patent patent application application number number 2011213585, 2011213585, the entire the entire disclosures disclosures of which of which are are incorporated herein incorporated herein by by reference. reference.

FIELD OF FIELD OF INVENTION INVENTION genetically modified

[0001a] AA genetically

[0001a] modified mouse is provided mouse is provided that that expresses expresses antibodies antibodies having having aa common human common human variable/mouse variable/mouse constant constant lightlight chain chain associated associated with with diverse diverse humanhuman variable/mouseconstant variable/mouse constantheavy heavy chains. chains. A method A method for making for making human bispecific a humanabispecific antibody antibody

from human from humanvariable variableregion regiongene gene sequences sequences of B of B cells cells of the of the mouse mouse is provided. is provided.

BACKGROUND BACKGROUND

[0002] Antibodies

[0002] Antibodiestypically typically comprise compriseaahomodimeric homodimeric heavy heavy chainchain component, component, wherein wherein each each heavychain heavy chainmonomer monomer is associated is associated with with an an identical identical light chain. light chain. Antibodies Antibodies having a having a heterodimeric heavy heterodimeric heavy chain chain component component (e.g.,bispecific (e.g., bispecific antibodies) antibodies) are are desirable desirable asas therapeutic antibodies. therapeutic antibodies. ButBut making making bispecific bispecific antibodies antibodies havinghaving a suitable a suitable light light chain chain component that component that cancan satisfactorilyassociate satisfactorily associatewith witheach each of the of the heavy heavy chains chains of a bispecific of a bispecific

antibody has proved antibody has provedproblematic. problematic.

[0003] In

[0003] In one one approach, approach,a alight light chain chain might mightbebeselected selectedby by surveying surveying usage usage statistics statistics forfor all all

light chain light chain variable variable domains, identifying the domains, identifying the most frequently employed most frequently employedlight lightchain chainininhuman human antibodies, andpairing antibodies, and pairingthat thatlight lightchain chainin invitro vitrowith withthethe two two chainschains heavyheavy of differing of differing

specificity. specificity.

[0004] InIn another

[0004] anotherapproach, approach, a light a light chain chain might might be selected be selected by observing by observing light light chain chain sequences sequences in ina aphage phage display display library library (e.g.,a aphage (e.g., phage display display librarycomprising library comprising human human lightlight

chain variable region chain variable region sequences, e.g., aa human sequences, e.g., ScFvlibrary) human ScFv library) and and selecting selecting the the most most commonly used commonly used light light chain chain variable variable region region fromfrom the library. the library. The light The light chainchain can bethen be can then

tested ononthe tested thetwotwo different different heavy heavy chains chains of interest. of interest.

[0005] In

[0005] In another anotherapproach, approach, a lightchain a light chainmight might be be selected selected by assaying by assaying a phage a phage displaydisplay

library ofoflight library chain light variable chain sequences variable using the sequences using the heavy heavychain chainvariable variablesequences sequences of both of both

heavychains heavy chainsofofinterest interestasasprobes. probes. A light A light chain chain thatthat associates associates with with both both heavy heavy chain chain variable sequences variable sequences might be selected might be selected as as aa light light chain chain for for the the heavy heavy chains. chains.

1

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[0006] In

[0006] In another another approach, approach,a acandidate candidate lightchain light chainmight mightbebe alignedwith aligned withthethe heavy heavy chains' chains'

cognate light chains, cognate light chains, and andmodifications modificationsare aremade made in the in the light light chain chain to to more more closely closely match match

sequence characteristicscommon sequence characteristics common to the to the cognate cognate lightlight chains chains of both of both heavyheavy chains. chains. If the If the May chances chances ofofimmunogenicity immunogenicity needneed to be to be minimized, minimized, the modifications the modifications preferably preferably result inresult in

sequences thatarearepresent sequences that present in in known known humanhuman light chain light chain sequences, sequences, such such that that proteolytic proteolytic

processing is processing is unlikely unlikely to togenerate T cell generate aa T cellepitope epitope based based on parametersand on parameters andmethods methods known known

in the in art for the art for assessing the assessing the likelihood likelihood of of immunogenicity immunogenicity (i.e., (i.e., in silico in silico as as as well well wetas wet assays). assays).

la 1a

[0007] All of the above approaches rely on in vitro methods that subsume a number of a 13 Oct 2025

priori restraints, e.g., sequence identity, ability to associate with specific pre-selected heavy chains, etc. There is a need in the art for compositions and methods that do not rely on manipulating in vitro conditions, but that instead employ more biologically sensible approaches to making human epitope-binding proteins that include a common light chain.

[0007a] Unless the context requires otherwise, where the terms “comprise”, “comprises”, “comprised” or “comprising” are used in this specification (including the claims) they are to 2023202781

be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

[0007b] A reference herein to a patent document or other matter which is given as prior art is not to be taken as admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0008] FIG. 1 illustrates a targeting strategy for replacing endogenous mouse immunoglobulin light chain variable region gene segments with a human V1-39J5 gene region.

[0009] FIG. 2 illustrates a targeting strategy for replacing endogenous mouse immunoglobulin light chain variable region gene segments with a human V3-20J1 gene region.

[0010] FIG. 3 illustrates a targeting strategy for replacing endogenous mouse immunoglobulin light chain variable region gene segments with a human VpreB/J5 gene region.

SUMMARY

[0011] In one aspect, the present invention provides a targeting vector comprising: (i) a 5’ homology arm comprising a nucleotide sequence corresponding to a genomic target sequence that is upstream to a 5’-most mouse Vκ gene segment in an unmodified endogenous mouse kappa light chain locus; (ii) a selection marker; (iii) a Vκ promoter sequence, (iv) a single rearranged immunoglobulin light chain Vκ/Jκ sequence; and (v) a 3’ homology arm comprising a nucleotide sequence corresponding to a genomic target sequence that is downstream of an endogenous mouse Jκ5 gene segment in an unmodified endogenous mouse kappa light chain locus, wherein the 3’ homology arm comprises a mouse Cκ exon.

2a

[0011a] In another aspect, the present invention provides a host cell comprising:(a) a first 13 Oct 2025

nucleic acid sequence that encodes a first immunoglobulin heavy chain, the first nucleic acid sequence comprising a first human immunoglobulin heavy chain variable region sequence that was identified from a B cell of a first mouse, wherein the first mouse has been immunized with a first antigen of interest including a first epitope, wherein the first human immunoglobulin heavy chain variable region sequence encodes a first human immunoglobulin heavy chain variable domain that recognizes the first epitope, and wherein 2023202781

the first mouse comprises in its germline genome: (i) an engineered mouse immunoglobulin kappa light chain locus comprising: a Vκ promoter sequence, a single rearranged immunoglobulin light chain Vκ/Jκ sequence, and a mouse Cκ sequence, and (ii) an engineered mouse immunoglobulin heavy chain locus comprising: unrearranged human immunoglobulin heavy chain variable region gene segments operably linked to a mouse immunoglobulin heavy chain constant region sequence at the endogenous heavy chain loci; and (b) a second nucleic acid sequence that encodes an immunoglobulin light chain comprising an immunoglobulin kappa light chain variable region sequence comprising the single rearranged immunoglobulin light chain Vκ/Jκ sequence, or a somatically hypermutated version thereof.

[0011b] In another aspect, the present invention provides a method for making an antibody comprising: expressing in a single cell: (a) a first nucleic acid sequence that encodes a first immunoglobulin heavy chain, the first nucleic acid sequence comprising a first human immunoglobulin heavy chain variable region sequence that was identified from a B cell of a first mouse, and wherein the first mouse has been immunized with a first antigen of interest including a first epitope, wherein the first human immunoglobulin heavy chain variable region sequence encodes a first human immunoglobulin heavy chain variable domain that recognizes the first epitope, and wherein the first mouse comprises in its germline genome: (i) an engineered mouse immunoglobulin kappa light chain locus comprising: a Vκ promoter sequence, a single rearranged immunoglobulin light chain Vκ/Jκ sequence, and a mouse Cκ sequence, and (ii) an engineered mouse immunoglobulin heavy chain locus comprising: unrearranged human immunoglobulin heavy chain variable region gene segments operably linked to a mouse immunoglobulin heavy chain constant region sequence at the endogenous heavy chain loci; and (b) a second nucleic acid sequence that encodes an immunoglobulin light chain, the second nucleic acid sequence comprising an immunoglobulin kappa light chain variable region sequence comprising the single rearranged immunoglobulin light chain Vκ/Jκ sequence, or a somatically hypermutated version thereof; maintaining the cell under conditions sufficient to express the antibody; and isolating the antibody.

2a

2b

[0011c] In another aspect, the present invention provides a mouse embryonic stem (ES) 13 Oct 2025

cell that has been genetically modified so that it comprises in its genome: (a) an engineered mouse immunoglobulin kappa light chain locus comprising: a Vκ promoter sequence, a single rearranged Vκ/Jκ sequence; and a mouse Cκ sequence; and (b) an engineered mouse immunoglobulin heavy chain locus comprising: a plurality of human immunoglobulin heavy chain variable region gene segments operably linked to an endogenous mouse immunoglobulin heavy chain constant region, wherein the plurality of human immunoglobulin 2023202781

heavy chain variable region gene segments replaces endogenous mouse immunoglobulin heavy chain variable region gene segments at the endogenous mouse immunoglobulin heavy chain locus.

[0011d] Genetically modified mice that express human immunoglobulin heavy and light chain variable domains, wherein the mice have a limited light chain variable repertoire, are provided. A biological system for generating a human light chain variable domain that associates and expresses with a diverse repertoire of affinity-matured human heavy chain variable domains is provided. Methods for making binding proteins comprising immunoglobulin variable domains are provided, comprising immunizing mice that have a limited immunoglobulin light chain repertoire with an antigen of interest, and employing an immunoglobulin variable region gene sequence of the mouse in a binding protein that specifically binds the antigen of interest. Methods include methods for making human immunoglobulin heavy chain variable domains suitable for use in making multi-specific antigen-binding proteins.

[0012] Genetically engineered mice are provided that select suitable affinity-matured human immunoglobulin heavy chain variable domains derived from a repertoire of unrearranged human heavy chain variable region gene segments, wherein the affinity-matured human heavy chain variable domains associate and express with a single human light chain variable domain derived from one human light chain variable region gene segment. Genetically engineered mice that present a choice of two human light chain variable region gene segments are also provided.

2b

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[0013]Genetically engineeredmice

[0013] Genetically engineered miceare areprovided provided thatexpress that express a limitedrepertoire a limited repertoireofofhuman human light chain light variabledomains, chain variable domains,or aor a single single humanhuman light variable light chain chain variable domain, domain, from from a limited a limited repertoire of repertoire of human light chain human light chain variable variable region region gene gene segments. The segments. The mice mice areare genetically genetically

engineeredtotoinclude engineered include aa single single unrearranged unrearrangedhuman human light light chain chain variableregion variable regiongene gene segment(or segment (ortwo twohuman human lightchain light chainvariable variableregion regiongene gene segments) segments) that that rearranges rearranges to form to form a a rearranged human rearranged human lightchain light chainvariable variableregion regiongene gene(or(ortwo tworearranged rearrangedlight lightchain chainvariable variable region genes) region genes) that that express express a single a single light light chainchain (or express (or that that express either either or both or bothlight of two of two light chains). The chains). Therearranged rearrangedhuman human light light chain chain variabledomains variable domains are are capable capable of pairing of pairing with with a a plurality pluralityofof affinity-matured human affinity-matured humanheavy heavy chains chains selected by the selected by the mice, mice, wherein wherein the the heavy heavy chain variable chain variableregions regions specifically specifically bindbind different different epitopes. epitopes.

[0014]In

[0014] In one aspect, aa genetically one aspect, genetically modified mouseisisprovided modified mouse providedthat that comprises comprisesa asingle single human immunoglobulin human immunoglobulin light light chain chain variable(VL) variable (VL)region regiongene gene segment segment thatthat is capable is capable of of

rearranging and encoding rearranging and encodinga ahuman human VL domain VL domain of anofimmunoglobulin an immunoglobulin light chain. light chain. In another In another

aspect, aspect, the the mouse comprises mouse comprises no no more more thanthan two two human human VL segments VL gene gene segments that arethat are capable capable

of rearranging of and encoding rearranging and encodinga ahuman humanVL VL domain domain of anofimmunoglobulin an immunoglobulin light light chain. chain.

[0015]In

[0015] In one aspect, aa genetically one aspect, genetically modified mouse mouseisisprovided providedthat that comprises comprisesa asingle single rearranged (V/J) human rearranged (V/J) humanimmunoglobulin immunoglobulin light light chain chain variable variable (VL) (VL) regionsegment region segment (i.e.,a aV/J (i.e., V/J segment)that segment) that encodes encodesa ahuman human VL domain VL domain of anof an immunoglobulin immunoglobulin light chain. light chain. In another In another

aspect, the aspect, the mouse comprises mouse comprises no no more more thanthan two two rearranged rearranged humanhuman VLsegments VL gene gene segments that that are capable are of encoding capable of encodinga ahuman humanVL VL domain domain of anofimmunoglobulin an immunoglobulin light light chain. chain.

[0016]in

[0016] one embodiment, In one embodiment,thethe VL VL gene gene segment segment is a is a human human VK1-39JK5 Vk1-39JK5 gene or gene segment segment a or a human human 3-20J1 gene gene VK3-20JKI segment. segment. In embodiment, In one one embodiment, the mouse the mouse has both has both a human a human Vk1- V1

gene segment 39JK5 gene 39Jk5 segment and and aa human human3-20J1 gene gene V3-20JK1 segment. segment.

[0017]In

[0017] one embodiment, In one embodiment,thethe human human VL gene VL gene segment segment is operably is operably linkedlinked to a human to a human or or mouseleader mouse leadersequence. sequence. In one In one embodiment, embodiment, the leader the leader sequence sequence is a mouse is a mouse leader leader sequence.In Ina aspecific sequence. specific embodiment, embodiment,thethe mouse mouse leader leader sequence sequence is a mouse is a mouse V3-7 3-7 leader leader sequence. sequence.

[0018]In

[0018] one embodiment, In one embodiment,thethe VL VL gene gene segment segment is operably is operably linked linked to immunoglobulin to an an immunoglobulin promoter sequence. promoter sequence.In In oneone embodiment, embodiment, the promoter the promoter sequence sequence is a human is a human promoterpromoter

sequence.InIna aspecific sequence. specific embodiment, embodiment,thethe human human immunoglobulin immunoglobulin promoter promoter is a VK 3 -15 is a Vk3-15

promoter. promoter.

[0019]In

[0019] one embodiment, In one embodiment,thethe geneticallymodified genetically modifiedmouse mouse comprises comprises VL locus a VL alocus that that does does

not comprise not anendogenous comprise an endogenous mouse mouse VL segment VL gene gene segment that is that is capable capable of rearranging of rearranging to formto form an immunoglobulin an immunoglobulinlight lightchain chain gene, gene,wherein whereinthe theVLVL locus locus comprises comprises a single a single human human VL VL gene segment gene segment thatisiscapable that capableofofrearranging rearrangingtotoencode encode a VL a VL region region of of a lightchain a light chaingene. gene.InIn

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a specific specific embodiment, thehuman humanVL VL gene segment is a is a human Vx1-39JK5 gene segment 2023202781 04 May 2023

a embodiment, the gene segment human Vk1-39Jk5 gene segment

or aa human or human VK3-20JK1 genesegment. V3-20Jk1 gene segment.

[0020]In

[0020] In one embodiment, one embodiment, theVLVL the locus locus comprises comprises a leader a leader sequence sequence flanked flanked 5' (with 5' (with

respect to transcriptional respect to transcriptionaldirection of of direction thethe VL VL gene genesegment) segment) with with aa human immunoglobulin human immunoglobulin

promoter andflanked promoter and flanked3'3' with with aa human humanVLVL gene gene segment segment that that rearranges rearranges and encodes and encodes VL VL domainofofaa reverse domain reversechimeric chimericlight light chain chain comprising an endogenous comprising an endogenous mouse mouse lightlight chain chain

constant region constant region (CL). (CL). In In aa specific specific embodiment, theVLVLgene embodiment, the gene segment segment is at is at thethe mouse mouse

kappa VL locus, (K) VL kappa (k) locus, and and the the mouse mouseCLCL is is a a mouse mouse K CL. K CL.

[0021]In

[0021] In one embodiment, one embodiment, themouse the mouse comprises comprises a nonfunctional a nonfunctional lambda lambda () (k) immunoglobulin light chain immunoglobulin light chain locus. locus. In In aa specific specific embodiment, the2 klocus embodiment, the locuscomprises comprisesa a

deletion of deletion of one one or or more sequencesofofthe more sequences thelocus, locus,wherein whereinthe theone oneorormore more deletionsrenders deletions renders the k2 locus the locus incapable of rearranging incapable of rearranging to to form a light form a lightchain chaingene. gene. In In another another embodiment all or embodiment all or substantially all of substantially all of the the VL VLgene gene segments segments of the of 2 the locus locusk are are deleted. deleted.

[0022]In one embodiment,

[0022] In one embodiment,thethe VLVL locus locus of of thethemodified modifiedmouse mouse is aisKalocus, K locus, andand thethe K K locus locus

comprisesaamouse comprises mouse K intronicenhancer, K intronic enhancer,a mouse a mouse 3' enhancer, K 3'K enhancer, or both or both an intronic an intronic

enhancerand enhancer anda a3'3'enhancer. enhancer.

[0023]In

[0023] In one embodiment, one embodiment, mouse mouse makes makes a light a light chain chain thatthat comprises comprises a somatically a somatically mutated mutated

VL domain VL domainderived derivedfrom froma ahuman human VL gene VL gene segment. segment. In one In one embodiment, embodiment, the the light light chain chain comprises somaticallymutated comprises aasomatically mutatedVLVL domain domain derived derived fromfrom a human a human VLsegment, VL gene gene segment, and a and a

mouseK KCLCL mouse region.In Inone region. one embodiment, embodiment, the mouse the mouse doesexpress does not not express a 2 light light chain. a k chain.

[0024]In

[0024] one embodiment, In one embodiment,thethe geneticallymodified genetically modifiedmouse mouse is capable is capable of somatically of somatically

hypermutating thehuman hypermutating the humanVL VL region region sequence. sequence. In a In a specific specific embodiment, embodiment, the mouse the mouse

comprisesa acell comprises cell that that comprises rearrangedimmunoglobulin comprises aa rearranged immunoglobulin lightchain light chaingene gene derived derived from from

the human the humanVLVL gene gene segment segment thatthat is capable is capable of rearranging of rearranging and and encoding encoding a VL a VL domain, domain, and and the rearranged the rearranged immunoglobulin immunoglobulin lightchain light chaingene genecomprises comprises a somatically a somatically mutated mutated VL VL domain. domain.

[0025]In

[0025] In one embodiment, one embodiment, thethe mouse mouse comprises comprises a cell a cell thatthat expresses expresses a light a light chain chain

comprising aasomatically comprising somatically mutated mutatedhuman humanVL VL domain domain linked linked to a to a mouse mouse CL, wherein K CL,Kwherein the the light chain light chain associates associates with with aaheavy heavy chain chain comprising somatically mutated comprising aa somatically mutatedVHVH domain domain

derived from derived from aa human humanVHVH gene gene segment segment and wherein and wherein the heavy the heavy chain comprises chain comprises a mouse a mouse heavychain heavy chainconstant constantregion region(CH). (CH).

[0026]In

[0026] one embodiment, In one embodiment,thethe mouse mouse comprises comprises a replacement a replacement of endogenous of endogenous mouse VHmouse VH gene segments gene segments with with one one or or more more human human VH gene segments, VH gene segments, wherein wherein the the human VH gene human VH gene segmentsare segments areoperably operably linkedtotoa amouse linked mouseCH CH region region gene, gene, suchsuch that that the the mouse mouse rearranges rearranges

the the human human VHVH gene gene segments segments and expresses and expresses a reverse a reverse chimeric chimeric immunoglobulin immunoglobulin heavy heavy chain that chain thatcomprises comprisesa human a humanVH VHdomain domain and and aa mouse mouse CH. In one CH. In one embodiment, embodiment, 90-100% 90-100%

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of unrearranged mouseVH VH genegene segments are replaced with at least one unrearranged 2023202781 04 May 2023

of unrearranged mouse segments are replaced with at least one unrearranged

humanVHVH human gene gene segment. segment. In a In a specific specific embodiment, embodiment, all orallsubstantially or substantially all all of of the the

endogenous mouse endogenous mouse VH gene VH gene segments segments are replaced are replaced with at with at one least leastunrearranged one unrearranged human human VH genesegment. VH gene segment. In one In one embodiment, embodiment, the replacement the replacement is at is with with at least least 19,least 19, at at least 39,39, or or at at

least 80 least 80 or or 81 81 unrearranged human unrearranged human VH VH genegene segments. segments. In oneInembodiment, one embodiment, the the replacementisis with replacement with at at least least 12 12 functional functionalunrearranged humanVHVH unrearranged human gene gene segments, segments, at least at least

25 functional 25 functional unrearranged human unrearranged human VH VH genegene segments, segments, or at or at least least 43 functional 43 functional

unrearranged human unrearranged VHgene human VH genesegments. segments.InInone oneembodiment, embodiment,the the mouse mousecomprises comprisesaa replacementofofall replacement all mouse andJ Jsegments mouse D Dand segments with with at at leastoneone least unrearranged unrearranged human human D D segmentand segment andatatleast leastone oneunrearranged unrearranged human human J segment. J segment. In oneInembodiment, one embodiment, the at the at least least one unrearranged one unrearrangedhuman human D segment D segment is selected is selected from from D1-7,D1-7, D3-3, D3-3, D1-26, D1-26, D3-10,D3-10, D3-16, D3-16, D3- D3 22, D5-5, 22, D5-12, D6-6, D5-5, D5-12, D6-6, D6-13, D6-13,D7-27, D7-27,andand a combination a combination thereof. thereof. In one In one embodiment, embodiment, the the at at least one least one unrearranged human unrearranged human J segment J segment is selected is selected fromfrom J, J3, J1, J3, J4, J4, J6,J6, J5,J5, andand a a combinationthereof. combination thereof. InIn aa specific specific embodiment, theone embodiment, the oneorormore more human human VH gene VH gene segment segment is is selectedfrom selected froma 1-2, a 1-2, 1-8, 1-8, 1-24, 1-24, 2-5,2-5, 3-7, 3-7, 3-9, 3-9, 3-11,3-11, 3-13, 3-13, 3-15, 3-15, 3-20,3-30, 3-20, 3-23, 3-23,3-33, 3-30, 3-33, 3-48, 3-48, 4-31, 4-31, 4-39, 4-39, 4-59, 4-59, 5-51, 5-51, aa 6-1 6-1 human VHgene human VH gene segment, segment, and and a combination a combination thereof. thereof.

[0027]In

[0027] one embodiment, In one embodiment, the the mouse mouse comprises comprises a B cell a B cell thatthat expresses expresses a binding a binding protein protein

that specifically binds that specifically bindsananantigen antigen of interest, of interest, wherein wherein the binding the binding proteinprotein comprises comprises a light a light chain derived chain derivedfrom froma human Vk1-39/Jk5rearrangement a humanVcl-39/JK5 rearrangementoror a human a humanV3-20/JK1 V3-20/J1

rearrangement,and rearrangement, andwherein whereinthethe cellcomprises cell comprises a rearranged a rearranged immunoglobulin immunoglobulin heavyheavy chain chain

genederived gene derived from fromaarearrangement rearrangementof of human human genegene segments segments selected selected from a from a VH2-5, VH2-5, VH3- VH3 23, VH3-30, 23, VH4-39, VH3-30, VH 4-39,VH4-59, VH4-59,andand VH5-51 VH5-51 gene gene segment. segment. In one In one embodiment, embodiment, the the one or one or more humanVH more human VHgene genesegments segmentsare arerearranged rearranged with with aa human heavy chain human heavy chain JJ gene gene segment segment selected from selected from J1, J1, J3, J3, J4, J4, J5, J5,and and J6. J6. In In one one embodiment, theone embodiment, the one oror more more human human VH Jand VH and J genesegments gene segmentsareare rearranged rearranged with with a human a human D gene D gene segment segment selected selected fromD1-26, from D1-7, D1-7, D1-26, D3-3, D3-10, D3-3, D3-10, D3-16, D3-16,D3-22, D5-5,D5-12, D3-22,D5-5, D5-12, D6-6, D6-6, D6-13, D6-13, and and D7-27. In a In D7-27. a specific specific

embodiment,thethelight embodiment, light chain chain gene genehas has1,1,2,2,3,3, 4, 4, or or 55 or or more more somatic hypermutations. somatic hypermutations.

[0028]In

[0028] one embodiment, In one embodiment, thethe mouse mouse comprises comprises a B cell a B cell thatthat comprises comprises a rearranged a rearranged

immunoglobulinheavy immunoglobulin heavy chain chain variable variable region region gene gene sequence sequence comprising comprising VH,and a VH, aJH, JH,DHand DH genesegment gene segment selected selected from from VH VH 2-5 2-5 + JH1 + JH1 + D6-6, + D6-6, VH3-23 VH3-23 + JH4 + JH4 + + D3, VH3-23 D3, VH3-23 + JH4 + + JH4 +

D3-10, VH3-30 D3-10, VH3-30+ + JH1 JH1 + D6-6, + D6-6, VH3-30 VH3-30 +JH3 +JH3 VH3-30VH3-30 + D6-6, + D6-6, JH4 +VH3-30 + JH4 ++D1-7, D1-7, +VH3-30 JH4 + + JH4 +

D5-12, VH3-30+ + D5-12, VH3-30 JH4 JH4 + D6-13, + D6-13, VH3-30 VH3-30 + JH4+ +JH4 VH3-30VH3-30 + D6-6, D6-6, JH4 + VH3-30 + JH4 ++D7-27, D7-27, +VH3-30 +

JH5 JH5 ++D3-22, VH3-30 D3-22,VH3-30 + JH5 + JH5 + D6-6, + D6-6, VH3-30 VH3-30 + JH5++JH5 VH4-39VH4-39 + D7-27, D7-27, JH3 +VH4- + JH3 + +D1-26, D1-26, VH4 59 ++ JH3 59 JH3++D3-16, VH4-59 D3-16,VH4-59 + JH3 + JH3 + D3-22, + D3-22, VH4-59 VH4-59 + JH4 ++ JH4 VH5-51 VH5-51 + D3-16, D3-16, + JH3 + + JH3 D5-5, + D5-5, VH5-51 VH5-51 + + JH5 JH5 + D6-13, andand + D6-13, VH5-51 VH5-51 + JH6+ +JH6 + D3-16. D3-16. In a specific In a specific embodiment, embodiment, the B cell the B cell

expresses bindingprotein expresses a abinding protein comprising comprisinga ahuman human immunoglobulin immunoglobulin heavyheavy chain chain variable variable

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region region fused with aa mouse fused with heavychain mouse heavy chainconstant constant region,and region, and a human a human immunoglobulin immunoglobulin light light

chainvariable chain variableregion region fused fused withwith a mouse a mouse light constant light chain chain constant region. region.

[0029]In

[0029] Inone oneembodiment, embodiment, the thehuman human VL VL gene gene segment segment is isa ahuman VK1-39JK5 gene human V1-39JK5 gene

segment,and segment, andthe themouse mouse expresses expresses a reverse a reverse chimeric chimeric light light chain chain comprising comprising (i) (i) a VLa VL domainderived domain derivedfrom fromthe thehuman humanVL VL genegene segment segment and a(ii)mouse and (ii) a mouse CL; wherein CL; wherein the the light light chain is chain is associated associated with with aa reverse reverse chimeric chimeric heavy chain comprising heavy chain comprising(i) (i) aa mouse andand mouse CHCH (ii)a a (ii)

somatically mutated somatically mutated human humanVH VH domain domain derived derived from from a human a human VHsegment VH gene gene segment selected selected fromaa1-2, from 1-2,1-8, 1-8,1-24, 1-24,2-5, 2-5, 3-7, 3-7, 3-9, 3-9, 3-11, 3-11, 3-13, 3-13, 3-15,3-15, 3-20,3-20, 3-23, 3-23, 3-30,3-48, 3-30, 3-33, 3-33,4-31, 3-48, 4-31, 4-39, 4-39, 4-59, 5-51, 4-59, 5-51, and 6-1 human and 6-1 humanVHVH gene gene segment, segment, and aand a combination combination thereof. thereof. In oneIn one embodiment,the embodiment, themouse mouse expresses expresses a light a light chain chain that that is is somaticallymutated. somatically mutated.In In oneone

embodiment embodiment the the CL CL is is a a mouse mouse K CL. K CL.

[0030]In

[0030] Inone oneembodiment, embodiment, the thehuman human VL VL gene gene segment segment is isa ahuman human V3-20J1 gene 3-20J1 gene

segment, andthe segment, and themouse mouse expresses expresses a reverse a reverse chimeric chimeric light light chain chain comprising comprising (i) (i) a VL a VL

domain derivedfrom domain derived fromthe thehuman humanVL VL genegene segment, segment, and a(ii)mouse and (ii) a mouse CL; wherein CL; wherein the light the light

chain is associated chain is associated with with aa reverse reverse chimeric chimeric heavy chain comprising heavy chain comprising(i) (i) aa mouse CH,andand mouse CH, (ii) (ii)

aa somatically somatically mutated human mutated human VH VH derived derived fromfrom a human a human VH segment VH gene gene segment selectedselected from a from a

1-2, 2-5, 1-2, 2-5,3-7, 3-7,3-9, 3-9,3-11, 3-20, 3-11, 3-23, 3-20, 3-30, 3-23, 3-33, 3-30, 4-59, 3-33, andand 4-59, 5-51 human 5-51 human VH genesegment, VH gene segment, and combinationthereof. and aa combination thereof. InInone oneembodiment, embodiment,the the mouse mouse expresses expresses a light a light chainchain that that is is

somatically mutated. InIn one somatically mutated. oneembodiment embodimentthe the CL aismouse CL is a mouse K CL.x CL.

[0031]In

[0031] Inone oneembodiment, embodiment, the themouse mouse comprises comprisesboth botha ahuman Vk1-39Jk5 gene humanVK1-39JK5 gene segment segment

and humanVk3-20Jk1 and aa human genegene VK3-20JK1 segment, segment, andmouse and the the mouse expresses expresses chimeric chimeric a reverse a reverse light light chain comprising chain comprising (i) (i) aa VL VL domain derivedfrom domain derived froma ahuman human gene gene VK1-39JK5 Vk1-39Jk5 segment segment or a or a human3-20J1 human VK3-20JK1 gene segment, gene segment, and (ii) and (ii) a CL; a mouse mouse CL; the wherein wherein lightthe lightischain chain is associated associated

with aa reverse with chimeric heavy reverse chimeric heavychain chaincomprising comprising(i) (i) aa mouse mouseCH, andand CH, (ii) aa somatically (ii) somatically mutated human mutated human VH VH derived derived fromfrom a human a human VHsegment VH gene gene segment selectedselected from1-8, from a 1-2, a 1-2, 1-8, 1-24, 1-24,

2-5, 3-7, 3-9, 2-5, 3-7, 3-9, 3-11, 3-11,3-13, 3-13,3-15, 3-15, 3-20, 3-20, 3-23, 3-23, 3-30,3-30, 3-33,3-33, 3-48, 3-48, 4-31,4-39, 4-31, 4-39, 4-59,a 6-1 4-59, 5-51, 5-51, a 6-1 human human VHVH gene gene segment, segment, and aand a combination combination thereof. thereof. In oneInembodiment, one embodiment, the mouse the mouse

expressesa alight expresses light chain chain that that is issomatically somaticallymutated. mutated. InInone oneembodiment theCLCLisisaamouse embodiment the mouseK K

CL. CL.

[0032]In

[0032] Inone oneembodiment, embodiment, 90-100% of the 90-100% of theendogenous endogenous unrearranged unrearrangedmouse mouse VH gene VH gene

segments arereplaced segments are replacedwith withatatleast leastone oneunrearranged unrearranged human human VH gene VH gene segment. segment. In a In a specific embodiment, specific all or embodiment, all or substantially substantiallyall allof of thethe endogenous endogenous unrearranged mouse unrearranged mouse VH VH

genesegments gene segmentsareare replaced replaced with with at at leastone least oneunrearranged unrearranged human human VH segment. VH gene gene segment. In In oneembodiment, one embodiment, the replacement the replacement is least is with at with at 18,least 18, at at least 39,least 39, 80, at least at least or 81 80, or 81 unrearrangedhuman unrearranged human VH gene VH gene segments. segments. In one In one embodiment, embodiment, the replacement the replacement is with atis with at least least 12 12 functional functional unrearranged humanVHVH unrearranged human gene gene segments, segments, at least at least 25 functional 25 functional

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unrearranged human unrearranged human VH VH genegene segments, segments, or at or at least least 43 unrearranged 43 unrearranged human human VH gene VH gene

segments. segments.

[0033]In

[0033] In one embodiment,thethegenetically one embodiment, geneticallymodified modifiedmouse mouse is ais C57BL a C57BL strain, strain, in in a specific a specific

embodiment selected embodiment selected from from C57BL/A, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/An, C57BL/GrFa, C57BL/KaLwN,C57BL/6, C57BL/6, C57BL/6J, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/1OScSn, C57BL/1OCr, C57BL/10Cr, C57BL/Ola. C57BL/Ola. In aa specific In specificembodiment, the genetically embodiment, the genetically modified modified mouse mouse isis aa mix mixof of an an aforementioned aforementioned129129 strain and strain and an an aforementioned C57BL/6 aforementioned C57BL/6 strain.In Inanother strain. another specificembodiment, specific embodiment,the the mouse mouse is is mix of aa mix of aforementioned 129strains, aforementioned 129 strains, or or aa mix mix of of aforementioned strains. InIn aa specific BL/6strains. aforementioned BL/6 specific embodiment,the embodiment, the129 129strain strainofofthe the mix mix is is aa 129S6 129S6(129/SvEvTac) (129/SvEvTac) strain. strain.

[0034]In

[0034] In one embodiment,thethemouse one embodiment, mouse expresses expresses a reverse a reverse chimeric chimeric antibody antibody comprising comprising a a light chain light chainthat thatcomprises comprises aa mouse CLand mouse KK CL anda asomatically somaticallymutated mutated human human VL domain VL domain

derived derived from froma ahuman Vk1-39Jk5 gene human VK1-39JK5 gene segment or aa human segment or 3-20J1 genegene human V3-20JK1 segment, segment, and and

heavy chain aa heavy chain that that comprises comprisesa amouse mouse and and CH CH a somatically a somatically mutated mutated humanhuman VH domain VH domain

derived from aa human derived from humanVHVH gene gene segment segment selected selected from from a 1-2, 1-2, 1-8, a 1-8, 1-24,1-24, 2-5,2-5, 3-7,3-7, 3-9,3-9, 3-11, 3-11,

3-13, 3-13, 3-15, 3-15, 3-20, 3-20, 3-23, 3-23, 3-30, 3-30, 3-33, 3-33,3-48, 3-48,4-31, 4-31,4-39, 4-39,4-59, 4-59,5-51, and 5-51, anda a6-1 6-1human VHgene human VH gene segment, whereinthe segment, wherein themouse mouse does does not not express express a fully a fully mouse mouse antibody antibody and does and does not express not express

aa fully fullyhuman antibody. In human antibody. In one one embodiment the embodiment the mouse mouse comprises comprises a1 light a K light chain chain locus locus thatthat

comprises comprises aa replacement replacement ofofendogenous endogenous mouse mouse KK VL VL gene gene segments segments with with the thehuman human V1 1-

39J5 genesegment 39Jk5 gene segmentor or thethe human human V K 3-20J1 Vk3-20Jk1 gene segment, gene segment, and comprises and comprises a replacement a replacement

of of all alloror substantially all all substantially endogenous endogenousmouse mouse VH genesegments VH gene segments with with a complete a complete or or

substantially substantially complete repertoire of complete repertoire ofhuman VHgene human VH gene segments. segments.

[0035]In

[0035] In one aspect, aa mouse one aspect, mousecell cellis is provided provided that that is is isolated isolatedfrom fromaamouse as described mouse as described herein. In herein. In one embodiment,the one embodiment, thecell cell is is an an ES EScell. cell. In In one embodiment,thethe one embodiment, cellisis aa cell

lymphocyte. InInone lymphocyte. oneembodiment, embodiment,the the lymphocyte lymphocyte is a isB acell. B cell. In one In one embodiment, embodiment, the Bthe B cell cell expresses chimericheavy expresses aachimeric heavychain chaincomprising comprising a variable a variable domain domain derived derived fromfrom a human a human gene gene

segment; anda alight segment; and light chain chain derived derived from from aa rearranged rearrangedhuman human V1-39/J Vk1-39/J segment, segment, rearranged rearranged

humanVk3-20/J human VK3-20/J segment, segment, or aorcombination a combination thereof; thereof; wherein wherein the the heavy heavy chainchain variable variable

domainisis fused domain fused to to aa mouse mouseconstant constantregion regionand and thethe lightchain light chainvariable variable domain domainisisfused fusedtoto aa mouse mouse orora ahuman human constant constant region. region.

[0036]In

[0036] In one aspect, aa hybridoma one aspect, hybridomaisisprovided, provided,wherein whereinthe thehybridoma hybridomais is made made with with B cell a Bacell

of of aa mouse asdescribed mouse as describedherein. herein.InIna aspecific specific embodiment, embodiment,thethe B cellisis from B cell from aa mouse mouseas as

described herein that described herein that has has been beenimmunized immunized with with an an immunogen immunogen comprising comprising an epitope an epitope of of interest, and interest, andthe theB Bcell cellexpresses expresses a binding a binding protein protein that the that binds binds the epitope epitope of interest, of interest, the the binding protein binding protein has a somatically has a somatically mutated human mutated human VH VH domain domain and aand a mouse mouse CH, and hasand CH, a has a human VLdomain human VL domainderived derived from from aa human or aa human VK1-39JK5 or human Vk1-39Jk5 genegene VK3-20JK1 human 3-20J1 segment segment

and a mouse and a CL. mouse CL.

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[0037]In

[0037] In one aspect, aa mouse one aspect, mouseembryo embryo is provided, is provided, wherein wherein thethe embryo embryo comprises comprises a donor a donor

ES cell ES cell that that isisderived derivedfrom froma amouse as described mouse as described herein. herein.

[0038]in

[0038] one In one aspect, aspect, a targeting a targeting vector vector is provided, is provided, comprising, comprising, from from 5' to 5' 3' in to 3' in transcriptional direction transcriptional directionwith withreference to to reference thethe sequences sequences of ofthe the5'5' and and3'3' mouse mouse homology homology

arms of the arms of the vector, vector, aa 5'5'mouse homologyarm, mouse homology arm, a human a human or mouse or mouse immunoglobulin immunoglobulin promoter, promoter,

aa human or mouse human or leader sequence, mouse leader sequence, and and aa human LCVRgene human LCVR genesegment segmentselected selected from from aa humanVk1-39Jk5 human VK1-39JK5oror aa human humanV3-20Jk1 V3-20J1 gene gene segment, segment, andand a 3'mouse a 3' mousehomology homology arm.In In arm.

one embodiment, one embodiment, thethe 5' 5' and3'3'homology and homology arms arms target target the the vector vector to to a sequence a sequence 5' with 5' with

respect to respect to an an enhancer sequence enhancer sequence that that is ispresent present5'5'and andproximal proximaltotothe themouse mouseK K constant constant

region gene. region gene. InIn one oneembodiment, embodiment,thethe promoter promoter is aishuman a human immunoglobulin immunoglobulin variable variable regionregion

gene segment gene segment promoter. promoter. In aIn specific a specificembodiment, embodiment, the the promoter promoter is a is a human human 3-15 V3-15 promoter. InIn one promoter. oneembodiment, embodiment,thethe leader leader sequence sequence is a ismouse a mouse leader leader sequence. sequence. In a In a specific specific embodiment, themouse embodiment, the mouse leader leader sequence sequence is aismouse a mouse leader leader V3-7 VK3-7 sequence. sequence.

[0039]In

[0039] one In one aspect, aspect, a targeting a targeting vector vector is provided is provided as described as described above, butabove, in placebut of in theplace of the 5' 5' mouse homology mouse homology armarm thethe human human or mouse or mouse promoter promoter is flanked is flanked 5'with 5' with a site-specific a site-specific

recombinaserecognition recombinase recognitionsite site(SRRS), (SRRS),andand in in placeofofthe place the3'3' mouse mouse homology homology arm arm the the humanLCVR human genegene LCVR segment segment is flanked is flanked 3'with 3' with an SRRS. an SRRS.

[0040]In

[0040] In one aspect, aa reverse one aspect, reverse chimeric chimeric antibody antibodymade madeby by a mouse a mouse as described as described herein, herein,

wherein the wherein the reverse reversechimeric chimericantibody antibodycomprises comprises a lightchain a light chaincomprising comprisinga amouse mouse CL and CL and a a human VL, human VL, and and aa heavy heavy chain chain comprising comprisinga ahuman human VH VH and and aa mouse CH. mouse CH.

[0041]In

[0041] one aspect, In one aspect, aa method methodfor formaking makinganan antibody antibody is is provided,comprising provided, comprising expressing expressing

in aa single in singlecell cell(a)(a) a first VH gene a first sequence VH gene sequenceofofananimmunized mouseasasdescribed immunized mouse described herein herein

fused with fused with aa human humanCHCH gene gene sequence; sequence; (b) a(b)VLagene VL gene sequence sequence of an immunized of an immunized mouse asmouse as described herein fused described herein fused with with aa human humanCL CL gene gene sequence; sequence; and, and, (c) maintaining (c) maintaining the cell the cell under under

conditionssufficient conditions sufficienttotoexpress express a fully a fully human human antibody, antibody, and isolating and isolating the antibody. the antibody. In one In one embodiment, thecell embodiment, the cellcomprises comprisesa asecond second VH VH genegene sequence sequence of a second of a second immunized immunized

mouseasasdescribed mouse described herein herein fused fused with with a human a human CH gene CH gene sequence, sequence, the VH the first firstgene VH gene sequence encodes sequence encodes a VH a VH domain domain that that recognizes recognizes a first a first epitope, epitope, andand the the second second VH gene VH gene

sequence encodes sequence encodes a VH a VH domain domain that that recognizes recognizes a second a second epitope, epitope, wherein wherein the epitope the first first epitope and the second and the secondepitope epitopeare arenot notidentical. identical.

[0042]in

[0042] one aspect, In one aspect, aa method methodfor formaking makingan an epitope-binding epitope-binding proteinis isprovided, protein provided, comprising exposinga amouse comprising exposing mouseas as described described herein herein withwith an immunogen an immunogen that comprises that comprises an an epitope epitope ofofinterest, interest,maintaining maintainingthe the mouse mouse under conditions under conditions sufficientsufficient for the for the mouse to mouse to

generate an generate animmunoglobulin immunoglobulin molecule molecule thatthat specificallybinds specifically bindsthe theepitope epitopeofofinterest, interest, and and isolating the isolating theimmunoglobulin immunoglobulin molecule molecule that specifically that specifically binds binds the the ofepitope epitope of wherein interest; interest; wherein the epitope-binding the epitope-binding protein protein comprises heavychain comprises aaheavy chainthat thatcomprises comprises a somatically a somatically mutated mutated

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humanVHVH human andand a mouse a mouse CH, associated CH, associated with awith a light light chain chain comprising comprising a mouse a mouse CL and CL a and a humanVL human VLderived derived from from aa human human Vk1-39 JK5oror aa human VK1-39 J5 human3-20 VK3-20 JK1 gene J1 gene segment. segment.

[0043]In

[0043] In one aspect, aa cell one aspect, cell that thatexpresses expresses an epitope-binding protein an epitope-binding protein is is provided, provided, wherein wherein

the cell the cell comprises: comprises: (a) (a) aa human VLnucleotide human VL nucleotidesequence sequence encoding encoding a human a human VL domain VL domain

derived from derived from aahuman Vk1-39Jk5 or human VK1-39JK5 or aahuman human VK3-20J1 genesegment, V3-20Jk1 gene segment,wherein wherein the the human human VL nucleotide VL nucleotide sequence sequenceis isfused fused(directly (directly or or through linker) totoa ahuman through aa linker) immunoglobulin human immunoglobulin

light lightchain chainconstant constant domain cDNAsequence domain cDNA sequence (e.g., (e.g., a human a human K constant K constant domain domain DNA DNA

sequence);and, sequence); and,(b) (b) aa first first human VHnucleotide human VH nucleotidesequence sequence encoding encoding a human a human VH domain VH domain

derived from derived from aa first first human VHnucleotide human VH nucleotidesequence, sequence, wherein wherein thethe firsthuman first humanVH VH nucleotide nucleotide

sequenceisisfused sequence fused(directly (directly or or through through a linker) linker)toto a human a human immunoglobulin heavy immunoglobulin heavy chain chain

constant domain constant domaincDNA cDNA sequence; sequence; wherein wherein the epitope-binding the epitope-binding protein protein recognizes recognizes a first a first

epitope. In epitope. In one one embodiment, embodiment,thethe epitope-binding epitope-binding proteinbinds protein binds thefirst the first epitope epitope with with aa dissociation constant dissociation constant of of lower lower than than 10-6 10 M,M,lower than1010M,8 M, lowerthan lower lower than than 10 M, M, lower 10-9lower than than 1010M,M,lower 10¹ lowerthan than10¹¹ 10-11 M,M, or or lowerthan lower than10¹² 1012 M. M.

[0044]In

[0044] one embodiment, In one embodiment, the the cellcomprises cell comprises a second a second human human VH nucleotide VH nucleotide sequence sequence

encoding aa second encoding second human VH domain, human VH domain, wherein wherein the the second second human VHsequence human VH sequenceisis fused fused (directly ororthrough (directly througha alinker) to to linker) a human a humanimmunoglobulin heavychain immunoglobulin heavy chainconstant constantdomain domain cDNA cDNA

sequence,and sequence, andwherein wherein thethe second second human human VH domain VH domain does does not not specifically specifically recognize recognize the the first firstepitope (e.g., displays epitope (e.g., displaysa adissociation dissociation constant constant of, e.g., of, e.g., 10 M, M, 10 10 M, 10~6 10-M, M, 104 M, or higher), or higher),

and wherein and whereinthe theepitope-binding epitope-bindingprotein protein recognizes recognizesthe thefirst first epitope epitope and the second and the secondepitope, epitope, and wherein and whereinthe thefirst first and and the the second immunoglobulinheavy second immunoglobulin heavy chains chains eacheach associate associate with with an an identical light identical light chain of (a). chain of (a).

[0045]In

[0045] one embodiment, In one embodiment, thethe second second VH domain VH domain binds binds the second the second epitope epitope with awith a dissociation dissociation constant constant that that is islower lowerthan than10~6 M, lower 10 M, lower than than 10 M,M, 10-7 lower lower than than 10 10- M, lower M, lower

than 10-9M,M,lower than 10 lower than10¹1010 than M, lower M, lower thanthan 10¹¹10-11 M, orM,lower or lower thanthan 10¹² 10-12 M. M.

[0046]In

[0046] In one embodiment, one embodiment, thethe epitope-binding epitope-binding proteincomprises protein comprises a firstimmunoglobulin a first immunoglobulin heavy chainand heavy chain anda asecond second immunoglobulin immunoglobulin heavy heavy chain, chain, each each associated associated with with an an identical identical

light lightchain chainderived derivedfrom from aa human VLgene human VL gene segment segment selected selected fromfrom a human a human V1-39JK5 Vk1-39Jk5 or a or a human3-20J1 human VK3-20JK1 gene segment, gene segment, wherein wherein the firstthe first immunoglobulin immunoglobulin heavy heavy chain chain binds binds a first a first epitope with epitope with aa dissociation dissociation constant constant in inthe thenanomolar to picomolar nanomolar to range, the picomolar range, the second second immunoglobulinheavy immunoglobulin heavy chain chain binds binds a second a second epitope epitope withwith a dissociation a dissociation constant constant in the in the

nanomolar nanomolar to picomolar to picomolar range,range, the epitope the first first epitope and theand theepitope second second areepitope are nottheidentical, not identical, the first immunoglobulin first heavychain immunoglobulin heavy chaindoes doesnot notbind bindthe thesecond secondepitope epitope or or binds binds thesecond the second epitope with epitope with aa dissociation dissociation constant constant weaker thanthe weaker than the micromolar range(e.g., micromolarrange (e.g.,the the millimolar millimolar range), the range), the second immunoglobulin second immunoglobulin heavy heavy chain chain doesdoes not not bindbind the the firstepitope first epitopeororbinds bindsthe the first epitope first with aadissociation epitope with dissociation constant constant weaker weaker than than the the micromolar micromolar range range (e.g., the (e.g., the

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millimolar millimolar range), range), and and one or more one or of the more of the VL, the VH VL, the of the VH of the first firstimmunoglobulin heavy immunoglobulin heavy

chain, chain, and the VH and the of the VH of the second immunoglobulin second immunoglobulin heavy heavy chain, chain, are are somatically somatically mutated. mutated.

[0047]In

[0047] In one embodiment,thethe one embodiment, first immunoglobulin first immunoglobulinheavy heavy chain chain comprises comprises a protein a protein A- A binding residue, and binding residue, and the the second immunoglobulin second immunoglobulin heavy heavy chain chain lacks lacks the the protein protein A-binding A-binding

residue. residue.

[0048]In

[0048] In one embodiment,thethe one embodiment, cellisis selected cell selected from from CHO, CHO,COS, COS, 293,293, HeLa, HeLa, and aand a retinal retinal

cell cell expressing a viral expressing a viralnucleic nucleicacid sequence acid sequence (e.g., (e.g.,a aPERC.6 cell). PERC.6¹TMcell).

[0049]in

[0049] In one aspect, aa reverse one aspect, reverse chimeric chimeric antibody antibodyisis provided, provided, comprising comprisingaahuman humanVH VH and and

mouseheavy aa mouse heavy chain chain constant constant domain, domain, a human a human VLa and VL and a mouse mouse light constant light chain chain constant domain, whereinthe domain, wherein theantibody antibodyisismade madeby by a process a process that that comprises comprises immunizing immunizing a mouse a mouse as as described herein described herein with with an an immunogen immunogen comprising comprising an epitope, an epitope, and and the antibody the antibody specifically specifically

binds the epitope binds the of the epitope of the immunogen withwhich immunogen with which themouse the mouse was was immunized. immunized. In one In one

embodiment, theVLVLdomain embodiment, the domain is somatically is somatically mutated. mutated. In one In one embodiment embodiment the VHthe VH domain domain is is somatically mutated. InIn one somatically mutated. oneembodiment, embodiment, both both thethe VL VL domain domain andVHthedomain and the VH domain are are somatically mutated. InIn one somatically mutated. oneembodiment, embodiment,thethe VL VL is linkedtotoa amouse is linked mouse K constant K constant domain. domain.

[0050]In

[0050] In one aspect, aa mouse one aspect, mouseisisprovided, provided,comprising human comprisinghuman heavy heavy chainchain variable variable genegene

segments replacingall segments replacing all or or substantially substantially all allmouse mouse heavy chain variable heavy chain variable gene genesegments segmentsat at thethe endogenous mouse endogenous mouse locus; locus; no more no more than than onetwoor human one or two human light chain light chain variable variable gene gene

segments selectedfrom segments selected froma arearranged rearranged and and VK1-39/J Vk1-39/J a rearranged a rearranged VK3-20/J Vk3-20/J segment segment or a or a combinationthereof, combination thereof, replacing replacing all all mouse light chain mouse light chain variable variable gene gene segments; whereinthe segments; wherein the humanheavy human heavy chain chain variable variable gene gene segments segments are linked are linked to atomouse a mouse constant constant gene, gene, and and the the humanlight human light chain chain variable variable gene genesegment(s) segment(s)is islinked linkedto to aa human humanorormouse mouse constant constant gene. gene.

[0051]In

[0051] one In one aspect, aspect, a mouse a mouse ES cellES cell comprising comprising a replacement a replacement of all or substantially of all or substantially all all mouseheavy mouse heavy chain chain variablegene variable gene segments segments with with human human heavy heavy chain variable chain variable gene gene segments, andnonomore segments, and more than than oneone or two or two rearranged rearranged human human light light chainchain V/J segments, V/J segments,

wherein the wherein the human humanheavy heavy chain chain variable variable gene gene segments segments are linked are linked to a to a mouse mouse

immunoglobulinheavy immunoglobulin heavy chain chain constant constant gene, gene, and and the the human human lightlight chain chain V/J V/J segments segments are are linked to linked to aa mouse or human mouse or humanimmunoglobulin immunoglobulin light light chain chain constant constant gene. gene. In aInspecific a specific embodiment,the embodiment, thelight light chain chain constant constantgene geneisisaamouse mouse constant constant gene. gene.

[0052]In

[0052] In one aspect, an one aspect, an antigen-binding antigen-binding protein protein made madebyby a a mouse mouse as described as described herein herein is is provided. In provided. In aa specific specific embodiment, theantigen-binding embodiment, the antigen-bindingprotein proteincomprises comprisesa ahuman human immunoglobulin heavy immunoglobulin heavy chain chain variable variable region region fused fused witha amouse with mouse constant constant region, region, and and a a humanimmunoglobulin human immunoglobulin light light chain chain variableregion variable regionderived derived from from a VK1-39 a Vk1-39 gene gene segment segment or a or a VK3 - 2gene 3-20 0 gene segment, segment, wherein wherein the light the light chainchain constant constant region region is a is a mouse mouse constant constant region. region.

[0053]In

[0053] In one aspect, aa fully one aspect, fully human antigen-bindingprotein human antigen-binding protein made madefrom froman an immunoglobulin immunoglobulin

variable region variable region gene sequencefrom gene sequence from a mouse a mouse as described as described herein herein is provided, is provided, wherein wherein the the

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antigen-binding protein antigen-binding protein comprises fully human comprises a afully heavychain human heavy chaincomprising comprising a human a human variable variable

region derived region from aa sequence derived from sequenceofofa amouse mouseas as described described herein, herein, andand a fullyhuman a fully human light light

chain comprising chain comprising aaVK1-39 Vk1-39 orora a3-20 V3-20 variable variable region. region. In one In one embodiment, embodiment, the light the light chainchain

variable region variable region comprises onetotofive comprises one five somatic mutations. InIn one somatic mutations. oneembodiment, embodiment,thethe lightchain light chain variableregion variable regionisisa acognate cognate light light chain chain variable variable regionregion that that is is paired paired in a B in a Bof cell cell the of the mouse mouse with the with theheavy heavy chain chain variable variable region. region.

[0054]In

[0054] one embodiment, In one embodiment, thethe fullyhuman fully human antigen-binding antigen-binding protein protein comprises comprises a firstheavy a first heavy chain and chain and aa second secondheavy heavy chain,wherein chain, wherein thethe firstheavy first heavychain chainand andthe thesecond second heavy heavy chain chain

comprisenon-identical comprise non-identical variable variable regions regions independently independentlyderived derivedfrom froma amouse mouseas as described described

herein, and herein, and wherein eachofofthe wherein each the first first and and second heavychains second heavy chainsexpress express from from a host a host cell cell

associated with associated with aa human humanlight lightchain chain derived derived from froma aVk1-39 gene VK1-39gene segment segment or a or a V3-20 3-20 gene gene segment.InInone segment. oneembodiment, embodiment, the the firstheavy first heavy chain chain comprises comprises a firstheavy a first heavy chain chain variable variable

regionthat region thatspecifically specificallybinds binds a firstepitope a first epitope of of a firstantigen, a first antigen, andand the the second second heavy heavy chain chain comprisesa asecond comprises secondheavy heavy chain chain variable variable region region thatspecifically that specifically binds binds aa second secondepitope epitopeofofa a secondantigen. second antigen. InInaa specific specific embodiment, embodiment,thethefirst first antigen antigen and and the the second secondantigen antigenare are different. InIna aspecific different. specificembodiment, embodiment, the the first firstantigen and antigen andthe thesecond second antigen antigen are are the the same, same,

andthe and thefirst first epitope epitopeand and thethe second second epitope epitope are notare not identical; identical; in a specific in a specific embodiment, embodiment,

bindingofofthe binding thefirst first epitope epitopebybya first a firstmolecule molecule of the of the binding binding protein protein does does not notbinding block block of binding of the the second epitopebybya asecond second epitope secondmolecule molecule of of thethe binding binding protein. protein.

[0055] InIn one

[0055] oneaspect, aspect,a afully fully human bindingprotein human binding protein derived derivedfrom froma ahuman human immunoglobulin immunoglobulin

sequenceofofaamouse sequence mouseas as described described herein herein comprises comprises a first a first immunoglobulin immunoglobulin heavy heavy chainchain and and a second a immunoglobulin second immunoglobulin heavy heavy chain, chain, wherein wherein the the firstimmunoglobulin first immunoglobulin heavy heavy chain chain

comprises comprises a firstvariable a first variable region region thatthat is not is not identical identical to ato a variable variable region region of theof the second second

immunoglobulinheavy immunoglobulin heavy chain, chain, and and wherein wherein the the firstimmunoglobulin first immunoglobulin heavy heavy chain chain comprises comprises a a wild-type protein wild-type protein AA binding binding determinant, determinant, and the second and the secondheavy heavychain chain lacksa awild-type lacks wild-type protein AA binding protein binding determinant. In one determinant. In one embodiment, embodiment,thethe firstimmunoglobulin first immunoglobulin heavy heavy chain chain

binds protein binds protein AA under isolation conditions, under isolation conditions,and and the the second immunoglobulinheavy second immunoglobulin heavy chain chain does does

not bind not bind protein proteinA Aor or binds binds protein protein at least A atAleast 10-fold, 10-fold, a hundred-fold, a hundred-fold, or a thousand-fold or a thousand-fold

weakerthan weaker thanthe thefirst first immunoglobulin heavychain immunoglobulin heavy chainbinds bindsprotein proteinA Aunder under isolationconditions. isolation conditions. In aa specific In specificembodiment, the first embodiment, the first and andthe thesecond second heavy chainsare heavy chains areIgG1 IgG1isotypes, wherein isotypes,wherein the second the heavychain second heavy chaincomprises comprises a modification a modification selected selected from from 95R95R (EU (EU 435R), 435R), 96F 96F (EU (EU 436F), and 436F), and aa combination combinationthereof, thereof,and andwherein whereinthethe first heavy first heavychain chainlacks lackssuch suchmodification. modification.

[0056]In

[0056] one aspect, In one aspect, aa method methodfor formaking makinga abispecific bispecificantigen-binding antigen-bindingprotein protein is is provided, provided, comprisingexposing comprising exposing a first a first mouse mouse as described as described herein toherein a firsttoantigen a first ofantigen ofthat interest interest that comprisesa afirst comprises first epitope, epitope, exposing a second exposing a mouse second mouse as as described described herein herein to to a second a second

antigen of interest antigen of interestthat thatcomprises comprises aa second epitope, allowing second epitope, allowing the the first firstand andthe thesecond second mouse mouse

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to to each mountimmune each mount immune responses responses to the to the antigens antigens of interest,identifying of interest, identifyinginin the the first first mouse mouse a

first first human heavy human heavy chain chain variable variable regionregion that the that binds binds theepitope first first epitope of theantigen of the first first antigen of of interest, identifying interest, in in identifying thethe second mouse second mouse aa second humanheavy second human heavy chain chain variable variable region region that that

binds the binds the second epitopeofofthe second epitope the second secondantigen antigenofofinterest, interest, making first fully making aa first fullyhuman human heavy heavy

chaingene chain gene that that encodes encodes a first a first heavyheavy chain chain that the that binds binds theepitope first first epitope of the of the first first antigen antigen of of interest, making interest, making aa second fully human second fully heavychain human heavy chaingene gene thatencodes that encodes a second a second heavy heavy chainchain

that binds that bindsthe thesecond second epitope epitope ofsecond of the the second antigen antigen of interest, of interest, expressing expressing the first the first heavy heavy chainand chain andthethe second second heavyheavy chain chain in in athat a cell cellexpresses that expresses a single a single fully fully human human light chain light chain derived from derived from aa human humanVk1-39 VK1-39or or a human a human 3-20VW3-20 gene segment gene segment to form a to form a bispecific bispecific

antigen-binding antigen-binding protein, protein, andand isolating isolating the bispecific the bispecific antigen-binding antigen-binding protein.protein.

[0057]In

[0057] one embodiment, In one embodiment,thethefirst first antigen antigen and andthe the second secondantigen antigenare arenotnotidentical. identical.

[0058]In

[0058] In one embodiment,thethefirst one embodiment, first antigen antigen and and the the second secondantigen antigenareareidentical, identical, and andthe the first firstepitope epitopeand andthe thesecond second epitope epitope are are not not identical. identical.InInone oneembodiment, binding of embodiment, binding of the the first first heavy chainvariable heavy chain variable region region to the to the first first epitope epitope doesdoes not block not block bindingbinding of the second of the second

heavy chain heavy chain variable variable region region to to the the second epitope. second epitope.

[0059]In one

[0059] In one embodiment, embodiment, the antigen the first first antigen is selected is selected from a antigen from a soluble solubleandantigen a cell and a cell

surface antigen surface antigen (e.g., (e.g., aatumor tumor antigen), antigen),and and the the second antigen comprises second antigen comprisesa acell cell surface surface receptor. In receptor. In aa specific specificembodiment, the cell embodiment, the cell surface surface receptor receptor is isan an immunoglobulin receptor. immunoglobulin receptor.

In In aa specific specificembodiment, the immunoglobulin embodiment, the immunoglobulinreceptor receptorisisananFcFcreceptor. receptor.InInone one embodiment,the embodiment, thefirst first antigen and the antigen and the second secondantigen antigenare arethe thesame same cellsurface cell surfacereceptor, receptor, andbinding and bindingof of thethe firstheavy first heavy chain chain to the to the first first epitope epitope does does not binding not block block binding of the of the second second heavy chain heavy chain to to the the second secondepitope. epitope.

[0060]In

[0060] one embodiment, In one embodiment,thethe lightchain light chainvariable variable domain domainofofthe thelight light chain chain comprises to comprises 22to

5 somatic 5 mutations. InIn one somatic mutations. oneembodiment, embodiment,thethe lightchain light chainvariable variabledomain domainis is somatically a asomatically

mutated cognate mutated cognatelight light chain chain expressed expressedinina aBBcell cell of of the the first firstor or thethe second secondimmunized immunized

mousewith mouse witheither either the the first first ororthe second the second heavy heavy chain chain variable variable domain. domain.

[0061]In

[0061] In one embodiment, one embodiment, thethe first fully first fully human heavychain human heavy chainbears bearsananamino amino acid acid

modificationthat modification thatreduces reduces its its affinity affinity to to protein protein A, A, andand he second he second fully heavy fully human human heavy chain chain does notcomprise does not comprise a modification a modification that reduces that reduces its affinity its affinity to protein to protein A. A.

[0062]In

[0062] In one aspect, an one aspect, an antibody antibody oror aa bispecific bispecific antibody antibody comprising humanheavy comprising aa human heavy chain chain

variable variable domain made domain made in inaccordance accordance with with thethe invention invention is isprovided. provided.In Inanother another aspect, aspect, useuse

of of aa mouse asdescribed mouse as describedherein hereintotomake make a fullyhuman a fully human antibody antibody or or a fullyhuman a fully human bispecific bispecific

antibody antibody isisprovided. provided.

[0063]Any

[0063] of the Any of the embodiments embodiments andand aspects aspects described described herein herein canused can be be used in conjunction in conjunction

with one with another, unless one another, unless otherwise otherwiseindicated indicated or or apparent apparentfrom fromthe thecontext. context. Other Other

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embodiments willbecome embodiments will become apparent apparent to those to those skilled skilled in in theart the artfrom froma areview reviewofofthe the ensuing ensuing description. description.

DETAILED DESCRIPTION DETAILED DESCRIPTION

[0064]This

[0064] This invention invention is not is not limited limited to particular to particular methods, methods, and experimental and experimental conditions conditions

described, as described, as such such methods methods and and conditions conditions maymay vary. vary. It isalso It is alsototobebeunderstood understood that that the the

terminology usedherein terminology used hereinisis for for the the purpose of describing purpose of describing particular particular embodiments only,and embodiments only, andisis not intended not intendedtotobe be limiting,since limiting, since thethe scope scope ofpresent of the the present invention invention is defined is defined by the by the claims. claims.

[0065]Unless

[0065] definedotherwise, Unless defined otherwise,all all terms terms and andphrases phrasesused used herein herein include include themeanings the meanings that the terms that the termsandand phrases phrases have attained have attained in the in the art, art, unless unless the contrary the contrary is clearlyisindicated clearly indicated or or clearly apparent clearly apparent from the context from the in which context in which the the term term or or phrase is used. phrase is Although any used. Although any methodsand methods andmaterials materialssimilar similaroror equivalent equivalent to to those those described describedherein hereincan canbebeused usedin inthe the practiceorortesting practice testingofofthe thepresent present invention, invention, particular particular methods methods and materials and materials are now are now described. All described. All publications publications mentioned arehereby mentioned are herebyincorporated incorporatedbyby reference. reference.

[0066] Theterm

[0066] The term"antibody", "antibody", as asused usedherein, herein,includes includesimmunoglobulin immunoglobulin molecules molecules comprising comprising

four four polypeptide polypeptide chains, chains, two heavy (H) two heavy (H) chains chainsand andtwo twolight light (L) (L) chains inter-connected by chains inter-connected by disulfide bonds. disulfide Eachheavy bonds. Each heavychain chaincomprises comprises a heavy a heavy chain chain variable variable (VH)(VH) region region and and a a heavy chain heavy chainconstant constantregion region(CH). The (CH).The heavy heavy chain chain constant constant region region comprises comprises threethree

domains,CH1, domains, CH1,CH2CH2 and and CH3.CH3. Each Each light light chainchain comprises comprises a light a light chainchain variable variable (VL) (VL) region region

and aa light and light chain chain constant constant region region (CL). (CL). The The VH andVLVLregions VH and regionscan can be be furthersubdivided further subdividedinto into regions of hypervariability, regions of hypervariability, termed termedcomplementarity determining regions complementarity determining regions(CDR), (CDR),interspersed interspersed with regions with that are regions that are more conserved,termed more conserved, termedframework framework regions regions (FR). (FR). EachEach VHVLand VH and VL comprisesthree comprises threeCDRs CDRsandand four four FRs, FRs, arranged arranged fromfrom amino-terminus amino-terminus to carboxy-terminus to carboxy-terminus in in the following the following order: order: FR1, FR1, CDR1, FR2,CDR2, CDR1, FR2, CDR2, FR3,FR3, CDR3, CDR3, FR4 (heavy FR4 (heavy chainmay chain CDRs CDRs be may be abbreviated as abbreviated as HCDR1, HCDR2and HCDR1, HCDR2 andHCDR3; HCDR3; lightchain light chain CDRs maybebeabbreviated CDRsmay abbreviated as as LCDR1, LCDR2 LCDR1,LCDR2 and and LCDR3. LCDR3. The"high The term term affinity" "high affinity" antibody antibody refers refers to antoantibody an antibody that that has has a KD a with respect KD with respect to to its itstarget epitope target epitopeabout aboutofof 109 10 M or lower M or lower (e.g., (e.g.,about about 1 1x X10~9 M, 11 Xx 10 10 M, 10 0 M, 10 M, 11 xX 1011 10¹¹ M, M, or about 11xX 1012 or about 10¹² M). In one M). In embodiment,KDKD one embodiment, isismeasured measured by surface by surface

plasmon resonance, plasmon resonance, e.g., e.g.,BIACORETM; BIACORE in in anotherembodiment, another embodiment,KDKDisis measured measuredbybyELISA. ELISA.

[0067]The

[0067] Thephrase phrase"bispecific "bispecific antibody" antibody" includes includes an an antibody antibodycapable capableofofselectively selectively binding binding two or two or more more epitopes. epitopes. Bispecific antibodies generally Bispecific antibodies generally comprise comprisetwo twononidentical nonidenticalheavy heavy chains,with chains, witheach each heavy heavy chainchain specifically specifically binding binding a different a different epitope-either epitope-either on two on two different different molecules(e.g., molecules (e.g., different differentepitopes epitopes on on two two different differentimmunogens) or on immunogens) or on the the same samemolecule molecule (e.g., different (e.g., different epitopes epitopes onon the the same same immunogen). immunogen). If a bispecific If a bispecific antibody antibody is capable is of capable of selectivelybinding selectively bindingtwotwo different different epitopes epitopes (a first (a first epitope epitope and aand a second second epitope),epitope), the of the affinity affinity of the first heavy the first chainforforthethe heavy chain firstepitope first epitope willgenerally will generally beleast be at at least one one toortwo to two or three three or fouror four

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or more or moreorders orders of magnitude of magnitude lower lower than than the the affinity affinity of theheavy of the first first chain heavyforchain for the the second second epitope, epitope, and vice versa. and vice Epitopesspecifically versa. Epitopes specifically bound by the bound by the bispecific bispecific antibody antibody can be on can be on the the sameor or same a different a different target target (e.g., (e.g., on on the the samesame or a different or a different protein). protein). Bispecific Bispecific antibodies antibodies can can be made, be made,for for example, example,bybycombining combining heavy heavy chains chains thatthat recognize recognize different different epitopes epitopes of of thethe

sameimmunogen. same immunogen. For example, For example, nucleic nucleic acid acid sequences sequences encoding encoding heavyvariable heavy chain chain variable sequencesthat sequences thatrecognize recognizedifferent different epitopes epitopesofof the the same sameimmunogen immunogencan can be fused be fused to nucleic to nucleic

acid sequences acid encoding sequences encoding thethe same same or differentheavy or different heavy chain chain constant constant regions, regions, andand suchsuch

sequencescan sequences can be be expressed expressed in ain cell a cellthat thatexpresses expressesan an immunoglobulin immunoglobulin light light chain. chain. A A typical typical bispecific bispecificantibody antibodyhas hastwo twoheavy heavy chains chains each havingthree each having three heavy heavychain chainCDRs, CDRs, followed by (N-terminal followed by (N-terminal to to C-terminal) C-terminal) aa CH1 domain,a ahinge, CH1 domain, hinge,a aCH2 CH2 domain, domain, and and a CH3 a CH3

domain,and domain, andananimmunoglobulin immunoglobulin lightchain light chainthat thateither eitherdoes doesnot notconfer conferepitope-binding epitope-binding specificity but specificity but that that can canassociate associate withwith eacheach heavyheavy chain, chain, or that or canthat can associate associate with each with each heavy chain heavy chain and andthat thatcan canbind bindone oneorormore moreofofthe theepitopes epitopesbound bound by by thethe heavy heavy chain chain

epitope-binding regions, epitope-binding regions, or or that that can can associate associate with with each heavychain each heavy chainand andenable enable bindingoror binding

one or one or both both of of the the heavy chains to heavy chains to one one or or both both epitopes. epitopes.

[0068]The

[0068] The term term "cell" "cell" includes includes any that any cell cell that is suitable is suitable for expressing for expressing a recombinant a recombinant nucleic nucleic acid sequence. acid Cellsinclude sequence. Cells includethose thoseofofprokaryotes prokaryotesand and eukaryotes eukaryotes (single-cellorormultiple- (single-cell multiple cell), bacterial cell), bacterial cells cells (e.g., (e.g., strains strains of of E. coli, Bacilus E. coli, spp., Streptomyces Bacillus spp., Streptomycesspp.,spp., etc.), etc.),

mycobacteria mycobacteria cells, cells, fungal fungal cells, cells, yeast yeast cellscells (e.g., (e.g., S. cerevisiae, S. cerevisiae, S. pombe, S. pombe, P. pastoris, P. pastoris, P. P. methanolica, methanolica, etc.),plant etc.), plant cells,insect cells, insect cells cells (e.g.,SF-9, (e.g., SF-9, SF-21, SF-21, baculovirus-infected baculovirus-infected insect insect cells, Trichopusia cells, etc.), non-human ni, etc.), Trichoplusia ni, non-human animal animal cells,cells, human human cells, orcells, cell or cell fusions fusions such as, such for as, for example,hybridomas example, hybridomasor or quadromas. quadromas. In some In some embodiments, embodiments, theiscell the cell is a human, a human, monkey,monkey,

ape, hamster, ape, hamster, rat, rat, or or mouse cell. In mouse cell. In some embodiments, some embodiments, thethe cellisiseukaryotic cell eukaryoticand andisis selected from selected from the the following following cells: cells: CHO (e.g., CHO CHO (e.g., K1,DXB-11 CHO K1, CHO,CHO, DXB-11 Veggie-CHO), Veggie-CHO), COS COS (e.g., (e.g.,COS-7), COS-7), retinal retinalcell, cell,Vero, CV1, Vero, CV1,kidney kidney(e.g., HEK293, (e.g., HEK293,293 293 EBNA, MSR EBNA, MSR 293, 293, MDCK, MDCK,

HaK, BHK),HeLa, HaK, BHK), HeLa, HepG2, HepG2, W138, WI38, MRC MRC 5, 5, Colo205, Colo205, HBHL-60, HB 8065, 8065, (e.g., HL-60,BHK21), (e.g., BHK21), Jurkat, Jurkat,

Daudi, A431(epidermal), Daudi, A431 (epidermal),CV-1, CV-1,U937, U937, 3T3, 3T3, L cell,C127 L cell, C127cell, cell,SP2/0, SP2/0,NS-0, NS-0,MMT MMT 060562, 060562,

Sertoli cell, Sertoli cell, BRL BRL 3A3A cell,HT1080 cell, HT1080 cell,cell, myeloma myeloma cell, cell, cell, tumor tumorandcell, andline a cell a cell line from derived derived from an aforementioned an aforementionedcell. cell. InIn some someembodiments, embodiments, the the cellcell comprises comprises one one or more or more viralviral genes, genes,

e.g., aa retinal e.g., retinal cell cell that that expresses a viralgene expresses a viral gene (e.g., (e.g., a PER.C6 a PER.C6 cell).TM cell).

[0069] The

[0069] Thephrase phrase"complementarity "complementarity determining determining region," region," or or thethe term term "CDR," "CDR," includes includes an an aminoacid amino acid sequence sequence encoded encoded by aby a nucleic nucleic acidacid sequence sequence of anoforganism's an organism's immunoglobulin immunoglobulin

genesthat genes that normally normally (i.e., (i.e., inina wild-type animal) a wild-type appears animal) appearsbetween between two frameworkregions two framework regionsininaa variable region variable region of of aa light lightoror a heavy a heavychain chainofof ananimmunoglobulin immunoglobulin molecule (e.g., an molecule (e.g., an antibody antibody

or aa TT cell or cellreceptor). receptor).AACDR canbe CDR can beencoded encodedby,by, forforexample, example, a germline a germline sequence sequence or a or a rearranged oror unrearranged rearranged unrearrangedsequence, sequence, and, and, for for example, example, by abynaive a naive or aormature a mature B cell B cell or or a a cell. AA CDR T cell. T canbebesomatically CDR can somaticallymutated mutated (e.g.,vary (e.g., varyfrom froma asequence sequence encoded encoded in anin an

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animal's animal's germline), germline), humanized, and/ormodified humanized, and/or modifiedwith withamino amino acidsubstitutions, acid substitutions,additions, additions, or or deletions. In some deletions. In circumstances some circumstances (e.g.,for (e.g., for aa CDR3), CDR3),CDRs CDRscan can be encoded be encoded byortwo by two or more more

sequences (e.g., germline sequences (e.g., germline sequences) sequences) thatare that arenot notcontiguous contiguous (e.g.,inin an (e.g., an unrearranged unrearranged nucleic acidsequence) nucleic acid sequence) but contiguous but are are contiguous in a nucleic in a B cell B cell nucleic acid sequence, acid sequence, e.g., as thee.g., as the

result result of ofsplicing splicingor or connecting connectingthe sequences the sequences (e.g., (e.g.,V-D-J V-D-J recombination to form recombination to form a heavy heavy

chain CDR3). chain CDR3).

[0070]The

[0070] Theterm term"conservative," "conservative,"when whenused used to to describe describe a conservative a conservative amino amino acidacid

substitution, includessubstitution substitution, includes substitution of of an an amino amino acid residue acid residue by another by another amino acidamino acid residue residue

having sidechain having a aside chain R group R group with with similar similar chemical chemical properties properties (e.g.,orcharge (e.g., charge or hydrophobicity). hydrophobicity).

In general, In conservative general, aaconservative amino amino acid substitution acid substitution willsubstantially will not not substantially change change the the functional functional

properties properties ofofinterest interestofofa aprotein, protein,forforexample, example, the ability the ability of aof a variable variable region region to specifically to specifically

bind a target bind a target epitope epitope with withaadesired desired affinity. affinity.Examples Examples of ofgroups groups of of amino amino acids that that have have

side chains side chainswith withsimilar similar chemical chemical properties properties include include aliphatic aliphatic side such side chains chains such as as glycine, glycine, alanine, valine,leucine, alanine, valine, leucine,andand isoleucine; isoleucine; aliphatic-hydroxyl aliphatic-hydroxyl side chains side chains such as such serineas andserine and

threonine; amide-containing threonine; side chains amide-containing side chainssuch suchasasasparagine asparagineandand glutamine; glutamine; aromatic aromatic sideside

chains such chains such as asphenylalanine, phenylalanine,tyrosine, tyrosine, and and tryptophan; tryptophan; basic basicside sidechains chainssuch suchasaslysine, lysine, arginine, and arginine, andhistidine; histidine;acidic acidic side side chains chains suchsuch as aspartic as aspartic acid acid and and glutamic glutamic acid; and,acid; and, sulfur-containing side sulfur-containing side chains chains such as cysteine such as cysteine and methionine. Conservative and methionine. Conservativeamino amino acids acids

substitutiongroups substitution groups include, include, for for example, example, valine/leucine/isoleucine, valine/leucine/isoleucine phenylalanine/tyrosine, phenylalanine/tyrosine,

lysine/arginine, alanine/valine, lysine/arginine, alanine/valine,glutamate/aspartate, glutamate/aspartate,and and asparagine/glutamine. In some asparagine/glutamine. In some

embodiments,a conservative embodiments, a conservative amino amino acidacid substitution substitution cancan be be substitutionofofany substitution anynative native residue in residue in aa protein proteinwith withalanine, alanine,asasused used in, in,forfor example, example,alanine alaninescanning scanningmutagenesis. In mutagenesis. In

someembodiments, some embodiments, a conservative a conservative substitution substitution is is made made thatthat hashas a positive a positive value value in in the the

PAM250 log-likelihoodmatrix PAM250 log-likelihood matrixdisclosed disclosedinin Gonnet Gonnetetetal. (1992) Exhaustive al. (1992) ExhaustiveMatching Matching of of thethe Entire Protein Entire Protein Sequence Database, Sequence Database, Science Science 256:1443-45, 256:1443-45, hereby hereby incorporated incorporated by reference. by reference.

In some In embodiments, some embodiments, thethe substitutionisisaa moderately substitution moderatelyconservative conservative substitutionwherein substitution whereinthethe substitution has substitution has aa nonnegative value in nonnegative value in the the PAM250 log-likelihoodmatrix. PAM250 log-likelihood matrix.

[0071]in

[0071] In some embodiments, some embodiments, residue residue positions positions in an in an immunoglobulin immunoglobulin lightlight chain chain or heavy or heavy

chain differ bybyone chain differ one or ormore more conservative aminoacid conservative amino acidsubstitutions. substitutions. In In some someembodiments, embodiments, residue positions in an immunoglobulin light chain or functional fragment thereof residue positions in an immunoglobulin light chain or functional fragment thereof (e.g., a (e.g., a fragmentthat fragment thatallows allows expression expression and secretion and secretion from, from, e.g., a Be.g., B cell) cell)a are are not identical not identical to a lightto a light chain chain whose amino whose amino acidsequence acid sequence is listedherein, is listed herein,but butdiffers differs by by one one or or more moreconservative conservative amino acidsubstitutions. amino acid substitutions.

[0072]The

[0072] Thephrase phrase"epitope-binding "epitope-bindingprotein" protein"includes includesa aprotein protein having havingatat least least one one CDR CDRandand that is that is capable capable ofofselectively selectively recognizing recognizing an epitope, an epitope, e.g., e.g., is is capable capable of binding of binding an an epitope epitope with aa KD with that is KD that isatatabout aboutone micromolar or one micromolar or lower (e.g.,a aKD lower (e.g., KDthat is is that about 1 x1 10. about X 10M, M, 11XX10-7 10

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M, 1 Xx 10 M, 10-9M,M,11X x1010-9 M, M, 1 XI x10¹10M, 1M,X110¹¹ x 10-11 M,about M, or or about 1x 1012 1 X 10¹² M). M). Therapeutic Therapeutic epitope epitope-

bindingproteins binding proteins(e.g., (e.g.,therapeutic therapeutic antibodies) antibodies) frequently frequently requirerequire is that a KD a KD that is nanomolar in the in the nanomolar or the or the picomolar range. picomolar range. May phrase"functional Thephrase

[0073] The

[0073] fragment"includes "functionalfragment" fragmentsof ofepitope-binding includesfragments proteinsthat epitope-bindingproteins that canbebeexpressed, can expressed, secreted, secreted, and specifically and specifically bind tobind to an with an epitope epitope a KD with in the KD in the micromolar, a micromolar, nanomolar, nanomolar, or or picomolar picomolar range. range. Specific Specific recognition recognition includes includes having a KDhaving that isaat that inis KDleast at least in the the micromolar range,the micromolar range, thenanomolar nanomolar range, range, or or thepicomolar the picomolar range. range.

[0074] The

[0074] Theterm term"germline" "germline"includes includesreference referencetotoananimmunoglobulin immunoglobulin nucleic nucleic acid acid sequence sequence

in aa non-somatically in non-somatically mutated mutated cell,cell, e.g.,e.g., a non-somatically a non-somatically mutated mutated cell cell B cell or Bpre-B or pre-B or cell or hematopoietic hematopoietic cell. cell.

[0075] Thephrase

[0075] The phrase"heavy "heavy chain,"oror"immunoglobulin chain," "immunoglobulin heavy heavy chain" chain" includes includes an an immunoglobulinheavy immunoglobulin heavy chain chain constant constant region region sequence sequence from from any organism. any organism. Heavy Heavy chain chain variable domains variable includethree domains include three heavy heavychain chainCDRs and and CDRs fourfour FR regions, FR regions, unless unless otherwise otherwise

specified. Fragments specified. Fragmentsofofheavy heavychains chains includeCDRs, include CDRs, CDRsCDRs and and and FRs, FRs,combinations and combinations thereof. AA typical thereof. typical heavy chain has, heavy chain has, following following the the variable variable domain (from N-terminal domain (from N-terminal to to C- C terminal), terminal), aa CH1 domain,a ahinge, CH1 domain, hinge,aaCH2 CH2domain, domain, andand a CH3 a CH3 domain. domain. A functional A functional fragment fragment

of aa heavy of heavychain chain includes includes a fragment a fragment that isthat is capable capable of specifically of specifically recognizing recognizing an epitope an epitope (e.g., recognizing (e.g., theepitope recognizing the epitope withwith a KDa in in micromolar, KDthe the micromolar, nanomolar, nanomolar, or picomolar or picomolar range), range), that is that is capable capable ofof expressing expressing and secreting and secreting from a from cell, cell, aand thatand that comprises comprises at at least one least one CDR. CDR.

[0076]The

[0076] Theterm term"identity" "identity" when usedinin connection when used connectionwith withsequence, sequence, includes includes identityasas identity

determinedbybya anumber determined numberof of differentalgorithms different algorithmsknown knownin inthe theart artthat that can can be beused usedtoto measurenucleotide measure nucleotideand/or and/oramino amino acid acid sequence sequence identity. identity. In some In some embodiments embodiments described described

herein, identities herein, identitiesare aredetermined determined using using aa ClustalW v. 1.83 ClustalW V. 1.83 (slow) (slow) alignment alignment employing an employing an

open gap open gappenalty penaltyofof10.0, 10.0, an an extend extendgap gappenalty penaltyofof0.1, 0.1, and andusing usingaaGonnet Gonnet similarity matrix similarity matrix TM 10.0.2, (MacVector10.0.2, MacVector Inc.,Inc., (MacVector MacVector 2008). 2008). The length The length of sequences of the the sequences compared compared with with respect to respect to identity identityofof sequences sequences will willdepend depend upon the particular upon the particular sequences, but in sequences, but in the the case case

of aa light of light chain constantdomain, chain constant domain, the length the length shouldshould containcontain sequencesequence of length of sufficient sufficient to length to fold into fold into aa light light chain constant chain constant domain domain that that is capable is capable of self-association of self-association to form to form a canonical a canonical

light chain light chainconstant constant domain, e.g., capable domain, e.g., capable of of forming forming two two beta beta sheets comprising beta sheets comprising beta strands and strands and capable capableofofinteracting interacting with with at at least leastone one CH1 domainofofaa human CH1 domain humanor or a mouse. a mouse. In In the case the of aa CH1 case of domain,the CH1 domain, thelength lengthofofsequence sequence should should contain contain sequence sequence of sufficient of sufficient

length to length to fold foldinto a aCH1 into CH1 domain that is domain that is capable capable of of forming forming two two beta beta sheets comprising beta sheets comprising beta strands and capable strands and capableofofinteracting interacting with with at at least leastone one light lightchain constant chain constantdomain domain of ofa amouse mouse

or or aa human. human.

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[0077]The

[0077] Thephrase phrase"immunoglobulin "immunoglobulin molecule" molecule" includes includes two two immunoglobulin immunoglobulin heavy heavy chainschains

and two and two immunoglobulin immunoglobulinlight lightchains. chains.The The heavy heavy chains chains may may be identical be identical or different,and or different, and the light chains the light may chains may be identical be identical or different. or different.

[0078]The

[0078] phrase"light The phrase "light chain" chain" includes includes an immunoglobulinlight an immunoglobulin lightchain chain sequence sequence from from anyany

organism, andunless organism, and unlessotherwise otherwisespecified specifiedincludes includeshuman human K and K and .light 2 light chains chains andand a VpreB, a VpreB,

as well as well asassurrogate surrogate light light chains. chains. LightLight chainchain variable variable (VL) domains (VL) domains typically typically include include three three light chain light chainCDRs andfour CDRs and four framework framework(FR) (FR)regions, regions,unless unless otherwise otherwise specified.Generally, specified. Generally, a a full-length light full-length light chain includes,from chain includes, from amino amino terminus terminus to carboxyl to carboxyl terminus, terminus, a VL a VL domain thatdomain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a and a light light chain chain constant constant domain.Light domain. Light chains include chains include those, those, e.g., e.g., that that do do not not selectively selectively bind bind eithereither a first a first or a or a second second epitope epitope

selectively selectively bound by the bound by the epitope-binding protein in epitope-binding protein in which which they they appear. Light chains appear. Light chains also also include those include that bind those that bind and recognize, or and recognize, or assist assistthe theheavy heavy chain chain with with binding binding and and

recognizing, one recognizing, one or or more moreepitopes epitopesselectively selectively bound boundbybythe theepitope-binding epitope-bindingprotein proteininin which which they appear. they appear. Common Common light light chains chains are are those those derived derived fromfrom a human a human V1-39JK5 Vk1-39Jk5 gene gene segment segment orora ahuman human V3-2OJK1 3-20J1 gene segment, gene segment, andsomatically and include include somatically mutated (e.g., mutated (e.g.,

affinity affinity matured) versions matured) versions of of thethe same. same.

[0079]The

[0079] Thephrase phrase"micromolar "micromolar range" range" is is intended intended to to mean mean 1-999 1-999 micromolar; micromolar; the phrase the phrase

"nanomolar range"isisintended "nanomolar range" intendedtoto mean mean1-999 1-999 nanomolar; nanomolar; the the phrase phrase "picomolar "picomolar range" range" is is intended to intended to mean mean1-999 1-999picomolar. picomolar.

[0080]The

[0080] Thephrase phrase"somatically "somaticallymutated" mutated"includes includes reference reference to to a a nucleicacid nucleic acidsequence sequence from from

cell that aa BB cell thathas hasundergone class-switching, wherein undergone class-switching, the nucleic wherein the nucleic acid acid sequence sequenceofofanan immunoglobulinvariable immunoglobulin variableregion region(e.g., (e.g., aa heavy chain variable heavy chain variable domain domainororincluding includingaaheavy heavy chain CDR chain CDR or sequence) or FR FR sequence) in the class-switched in the class-switched B cell B cell is not is not to identical identical to the the nucleic nucleic acid acid

sequence sequence in the in the B cell B cell prior prior to class-switching, to class-switching, such such as, foras, for example, example, a difference a difference in a CDR in a CDR or or framework nucleicacid framework nucleic acid sequence sequence between between B cell a B acell thathashas that notnot undergone undergone class class-

switching and switching and aa BB cell cell that that has has undergone class-switching. "Somatically undergone class-switching. "Somaticallymutated" mutated"includes includes referencetotonucleic reference nucleic acid acid sequences sequences from affinity-matured from affinity-matured B cells B cells that thatidentical are not are not to identical to correspondingimmunoglobulin corresponding immunoglobulin variable variable region region sequences sequences in B incells B cells that that areare notnot affinity affinity-

matured (i.e., sequences matured (i.e., in the sequences in the genome genomeofofgermline germlinecells). cells). The Thephrase phrase "somatically "somatically

mutated" also mutated" also includes includes reference referenceto to an an immunoglobulin immunoglobulinvariable variableregion regionnucleic nucleicacid acid sequence sequence from from a B cell a B cell afterafter exposure exposure of the of the B B cell to cell to an epitope an epitope of interest, of interest, wherein wherein the the nucleic acid nucleic acid sequence differs from sequence differs from the the corresponding nucleicacid corresponding nucleic acid sequence sequence priortoto prior

exposure exposure of of thethe B cell B cell to the to the epitope epitope of interest. of interest. The phrase The phrase "somatically "somatically mutated" mutated" refers to refers to sequencesfrom sequences fromantibodies antibodies thathave that have been been generated generated in animal, in an an animal, e.g., e.g., a mouse a mouse having having

humanimmunoglobulin human immunoglobulin variable variable region region nucleic nucleic acid acid sequences, sequences, in response in response to anto an

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immunogen challenge, immunogen challenge, andand that that resultfrom result fromthetheselection selectionprocesses processesinherently operativein in inherentlyoperative such an animal. such an animal.

[0081]The

[0081] term"unrearranged," The term "unrearranged,"with withreference referencetotoa anucleic nucleicacid acidsequence, sequence,includes includes nucleic nucleic

acid sequences acid sequences thatthat exist exist in the in the germline germline of an animal of an animal cell. cell.

[0082] Thephrase

[0082] The phrase"variable "variabledomain" domain"includes includesanan amino amino acid acid sequence sequence of anof an

immunoglobulin light or immunoglobulin light or heavy heavy chain chain(modified (modified asasdesired) desired) that that comprises comprisesthe thefollowing following amino acid amino acid regions, regions, in in sequence fromN-terminal sequence from N-terminaltotoC-terminal C-terminal(unless (unlessotherwise otherwiseindicated): indicated): FR1, FR1, CDR1, FR2, CDR2, CDR1, FR2, CDR2,FR3, FR3,CDR3, CDR3,FR4. FR4.

CommonLight Common LightChain Chain

[0083] Priorefforts

[0083] Prior effortstotomake make useful useful multispecific multispecific epitope-binding epitope-binding proteins, proteins, e.g., bispecific e.g., bispecific

antibodies, have antibodies, beenhindered have been hinderedbybyvariety varietyofof problems problemsthat thatfrequently frequently share sharea acommon common paradigm:in invitro paradigm: vitroselection selection or or manipulation manipulation of sequences of sequences to rationally to rationally engineer,engineer, or to or to engineerthrough engineer through trial-and-error, trial-and-error, a suitable a suitable format format for pairing for pairing a heterodimeric a heterodimeric bispecific bispecific

humanimmunoglobulin. human immunoglobulin. Unfortunately, Unfortunately, mostmost if not if not allallofofthe theinin vitro vitro engineering engineering approaches approaches

providelargely provide largelyadad hochoc fixes fixes thatthat are are suitable, suitable, if atif all, at all, forfor individual individual molecules. molecules. On theOn the other other hand, in hand, in vivo vivo methods for employing methods for employingcomplex complex organisms organisms to select to select appropriate appropriate pairings pairings that that

are capable are capable of of leading leading to to human therapeuticshave human therapeutics have notbeen not been realized. realized.

[0084] Generally,

[0084] Generally, native native mouse mousesequences sequences are are frequently frequently notnot a good a good source source for for human human

therapeutic sequences.ForForat atleast therapeutic sequences. leastthat that reason, reason, generating generatingmouse mouse heavy heavy chain chain

immunoglobulinvariable immunoglobulin variableregions regionsthat that pair pair with with aa common human common human light light chain chain is is of of limited limited

practical utility. practical utility. More More ininvitro vitro engineering engineering efforts efforts would would be expended be expended in a trial-and-error in a trial-and-error

process to process to try try totohumanize the mouse humanize the mouseheavy heavy chain chain variable variable sequences sequences while while hoping hoping to retain to retain

epitopespecificity epitope specificityand and affinitywhile affinity while maintaining maintaining the ability the ability to couple to couple withcommon with the the common human human light chain, light chain,with withuncertain uncertainoutcome. outcome. At At the the end of such end of process, the such a process, the final finalproduct product may may

maintainsome maintain some of the of the specificity specificity and affinity, and affinity, and associate and associate with with the the light common common chain,light but chain, but ultimately ultimately immunogenicity in aa human immunogenicity in humanwould would likelyremain likely remaina aprofound profound risk. risk.

[0085]Therefore,

[0085] Therefore, aa suitable suitable mouse mousefor formaking makinghuman human therapeutics therapeutics would would include include a suitably a suitably

large large repertoire repertoire of ofhuman heavychain human heavy chainvariable variableregion region gene genesegments segments in place in place of of

endogenous endogenous mouse mouse heavy heavy chainchain variable variable region region gene gene segments. segments. Theheavy The human human heavy chain chain variable region variable region gene segmentsshould gene segments should be be able able to to rearrange rearrange andand recombine recombine with with an an endogenous endogenous mouse mouse heavy heavy chainchain constant constant domain domain to aform to form a reverse reverse chimeric chimeric heavy heavy chain chain (i.e., (i.e., a aheavy heavychain chaincomprising comprising aa human variable domain human variable domainand and a mouse a mouse constant constant region). region).

The heavy The heavychain chainshould shouldbebe capable capable of of class class switching switching and and somatic somatic hypermutation hypermutation so that so that a a suitably large suitably largerepertoire repertoireof of heavy heavy chain chain variable variable domains domains are available are available for the for the mouse to mouse to

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select one select onethat thatcancan associate associate with with the limited the limited repertoire repertoire of light of human human light chain chain variable variable regions. regions.

[0086]A mouse

[0086]. mouse that that selects selects a common a common lightlight chain chain for for a pluralityofofheavy a plurality heavychains chainshas has a a May practical utility. In practical utility. In various embodiments, various embodiments, antibodies antibodies that express that express in that in a mouse a mouse that can only can only

express common express aa common lightchain light chainwill will have haveheavy heavy chains chains thatcan that can associate associate andand express express withwith an an

identical or substantially identical or substantiallyidentical identicallight lightchain. chain.ThisThis is particularly is particularly useful useful in making in making bispecific bispecific

antibodies. For example, antibodies. For example,such sucha amouse mouse can can be immunized be immunized with awith a first first immunogen immunogen to to generate B cell generate a Ba cell that that expresses expresses an antibody an antibody that specifically that specifically binds a binds a first epitope. first epitope. The The mouse (oraamouse mouse (or mouse geneticallythe genetically thesame) same) cancan be immunized be immunized with with a second a second immunogen immunogen to to generate B cell a cell generate a B that that expresses expresses an antibody an antibody that specifically that specifically binds binds the theepitope. second second epitope. Variable heavy Variable heavyregions regionscan canbebecloned clonedfrom fromthetheB B cellsand cells andexpresses expresses with with thethe same same heavy heavy

chain constant region, chain constant region, and and the the same samelight light chain, chain, and and expressed expressedininaacell cell to to make bispecific make aa bispecific

antibody, antibody, wherein the light wherein the light chain chain component component ofofthe the bispecific bispecific antibody antibody has beenselected has been selectedbyby aa mouse mouse totoassociate associateand andexpress express with with thelight the chain component. light chain component.

[0087]The

[0087] Theinventors inventorshave haveengineered engineered a mouse a mouse for generating for generating immunoglobulin immunoglobulin light light chains chains

that will that will suitably pair with suitably pair with aa rather ratherdiverse diverse family family of of heavy heavy chains, chains, including including heavy heavy chains chains whosevariable whose variableregions regionsdepart departfrom fromgermline germlinesequences, sequences, e.g., e.g., affinity matured affinity maturedororsomatically somatically mutatedvariable mutated variable regions. regions. InIn various various embodiments, embodiments, thethe mouse mouse is devised is devised to pair to pair human human lightlight

chain variable domains chain variable with human domains with human heavy heavy chain chain variable variable domains domains thatthat comprise comprise somatic somatic

mutations,thus mutations, thus enabling enabling a route a route to high to high affinity affinity binding binding proteins proteins suitable suitable for use for use as as human human therapeutics. therapeutics.

[0088]The

[0088] Thegenetically genetically engineered engineeredmouse, mouse, through through thethe long long andand complex complex process process of of antibody selection antibody selection within within an an organism, makesbiologically organism, makes biologically appropriate appropriate choices choicesinin pairing pairing aa diverse collection ofofhuman diverse collection heavychain human heavy chainvariable variabledomains domains witha alimited with limitednumber numberof of human human

light chain light options.In Inorder chain options. order to to achieve achieve this,this, the mouse the mouse is engineered is engineered toa present to present limited a limited numberofofhuman number human lightchain light chainvariable variabledomain domain options options in inconjunction conjunctionwith witha awide widediversity diversityofof humanheavy human heavy chain chain variable variable domain domain options. options. UponUpon challenge challenge withimmunogen, with an an immunogen, the the mousemaximizes mouse maximizes thethe number number of solutions of solutions in itsrepertoire in its repertoiretoto develop developananantibody antibodytotothe the immunogen, immunogen, limited limited largely largely or solely or solely by theby the number number or light or light chain chaininoptions options in its repertoire. its repertoire. In In various embodiments, various embodiments,this thisincludes includesallowing allowingthe themouse mouseto to achieve achieve suitable suitable andand compatible compatible

somatic mutationsofofthe somatic mutations the light light chain chain variable variable domain that will domain that willnonetheless nonetheless be be compatible with compatible with

relatively large aa relatively largevariety varietyofofhuman human heavy heavy chain chain variable variable domains,domains, including including in particular in particular

somatically mutated human somatically mutated human heavy heavy chain chain variable variable domains. domains.

[0089]ToToachieve

[0089] achieve a limited a limited repertoire repertoire of light of light chainchain options, options, theismouse the mouse is engineered engineered to to render nonfunctional render nonfunctional or substantially or substantially nonfunctional nonfunctional its ability its ability to make, to make, or rearrange, or rearrange, a native a native

mouselight mouse light chain chain variable variable domain. domain.This Thiscan canbebe achieved, achieved, e.g.,bybydeleting e.g., deletingthe themouse's mouse's light light

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chain variable chain variable region region gene segments.TheThe gene segments. endogenous endogenous mousemouse locus locus can canbethen then be modified modified

by an by an exogenous exogenoussuitable suitablehuman human lightchain light chainvariable variableregion regiongene gene segment segment of choice, of choice,

operably linked operably linked to to the the endogenous mouse endogenous mouse lightchain light chainconstant constant domain, in in domain, a manner a manner suchsuch

that the that the exogenous human exogenous human variable variable region region gene gene segments segments can rearrange can rearrange and recombine and recombine

with the with the endogenous mouse endogenous mouse light light chain chain constant constant region region gene gene and and formform a rearranged a rearranged reverse reverse

chimeric light chimeric light chain chaingene gene (human variable, mouse (human variable, mouseconstant). variousembodiments, constant).In Invarious embodiments, the the light chain light variableregion chainvariable regionisis capable capableofof being somaticallymutated. somatically being mutated. In Invarious variousembodiments, embodiments,

to maximize to maximize abilityof ofthethelight ability lightchain chain variable variable region region to acquire to acquire somatic somatic mutations, mutations, the the enhancer(s) isis retained appropriate enhancer(s) appropriate retained in in the the mouse. Forexample, mouse. For example,in inmodifying modifyinga amouse mouse K x

locus to locus to replace replace endogenous mouse endogenous mouse K variable K variable region region gene gene segments segments with human with human K variable K variable

region gene region segments,the gene segments, themouse mouse K intronic K intronic enhancer enhancer andand mouse mouse K 3' K 3' enhancer enhancer are are functionallymaintained, functionally maintained,or or undisrupted. undisrupted.

[0090]A geneticallyengineered

[0090]A genetically engineeredmouse mouse is provided is provided that that expresses expresses a limited a limited repertoire repertoire of of

reverse chimeric (human reverse chimeric variable,mouse (humanvariable, mouse constant) constant) chainsassociated lightchains light associated witha adiversity with diversity of reverse chimeric of reverse chimeric (human mouseconstant) variable, mouse (human variable, constant)heavy heavy chains. chains. In various In various

embodiments,the embodiments, theendogenous endogenous mouse mouse K light K light chainchain variable variable region genegene region segments segments are are deleted and replaced deleted and replaced with with aa single single (or (or two) two) human light chain human light chain variable variable region region gene gene

segments, linkedtoto the operablylinked segments, operably the endogenous endogenous mouse mouse K constant K constant region region gene.gene. In In embodiments formaximizing embodiments for maximizing somatic somatic hypermutation hypermutation of the of the human human light light chainchain variable variable region region

gene segments,the gene segments, themouse mouse K intronic K intronic enhancer enhancer and and the the mouse mouse K 3' K 3' enhancer enhancer are are maintained. InIn various maintained. various embodiments, embodiments, thethe mouse mouse alsoalso comprises comprises a nonfunctional a nonfunctional k light 2 light

locus,orora adeletion chainlocus, chain deletion thereof thereof or aordeletion a deletion that that renders renders the unable the locus locus to unable make ato2 make a k light chain. light chain.

[0091]A

[0091] genetically genetically engineered engineered mouse mouse is provided is provided that, that, in various in various embodiments, embodiments, comprises comprises

a light a lightchain chainvariable variableregion regionlocus locuslacking lackingananendogenous mouse endogenous mouse lightchain light chainvariable variable gene gene segmentand segment andcomprising comprising a human a human variable variable genegene segment, segment, in oneinembodiment one embodiment a rearranged a rearranged

humanV/J human V/Jsequence, sequence, operably operably linked linked to to a mouse a mouse constant constant region, region, wherein wherein the locus the locus is is capable of capable of undergoing undergoingsomatic hypermutation, somatichypermutation, andand wherein wherein the the locus locus expresses expresses a light a light chain chain

the human comprising the comprising humanV/J V/Jsequence sequence linked linked a mouse to mouse to a constant constant region. region. Thus,Thus, in various in various

embodiments,thethelocus embodiments, locuscomprises comprises a mouse a mouse 3' enhancer, K 3'Kenhancer, whichwhich is correlated is correlated withwith a a normal,ororwild-type, normal, wild-type,level levelof of somatic somatic hypermutation. hypermutation.

[0092] The

[0092] Thegenetically genetically engineered engineeredmouse mousein in various various embodiments embodiments when when immunized immunized with an with an antigenofofinterest antigen interestgenerates generates thatthat B cells B cells exhibit exhibit a diversity a diversity of rearrangements of rearrangements of human of human immunoglobulinheavy immunoglobulin heavy chain chain variable variable regions regions thatexpress that andand express function function with with oneone or or withtwo with two light chains, rearranged light rearranged chains, including includingembodiments where embodiments where thethe oneone or or twotwo lightchains light comprise chainscomprise human human light light chain chain variable variable regions regions that comprise, that comprise, e.g., 1 e.g., 1 to 5 somatic to 5 somatic mutations.mutations. In various In various

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embodiments, thehuman embodiments, the human light light chains chains so so expressed expressed are are capable capable of associating of associating and and

expressing with any expressing with any human human immunoglobulin immunoglobulin heavy heavy chainchain variable variable region region expressed expressed in thein the

mouse. mouse.

Epitope-binding Proteins Epitope-binding Proteins Binding Binding More Than One More Than OneEpitope Epitope

[0093]The

[0093] compositionsandand The compositions methods methods of described of described herein herein can can be used be used to make to make binding binding

proteinsthat proteins thatbind bindmore more thanthan one epitope one epitope withaffinity, with high high affinity, e.g., bispecific e.g., bispecific antibodies. antibodies.

Advantages Advantages of the of the invention invention include include the ability the ability to select to select suitably suitably high binding high binding (e.g., affinity (e.g., affinity

matured) heavy matured) heavychain chainimmunoglobulin immunoglobulin chains chains eacheach of which of which willwill associate associate with with a single a single light light

chain. chain.

[0094]Synthesis

[0094] Synthesisand andexpression expressionof of bispecificbinding bispecific binding proteins proteins has hasbeen beenproblematic, problematic,ininpart part due toissues due to issuesassociated associated with with identifying identifying a suitable a suitable light chain light chain that that can can associate associate and and express with two express with two different different heavy chains, and heavy chains, in part and in part due due to to isolation isolationissues. issues.The The methods methods

and compositionsdescribed and compositions described hereinallow herein allowfor foraagenetically genetically modified modified mouse mousetotoselect, select,through through otherwise natural otherwise natural processes, processes, aa suitable suitable light light chain chainthat thatcan canassociate associate and and express with more express with more

than one than oneheavy heavy chain, chain, including including heavy heavy chains chains that are that are somatically somatically mutated mutated (e.g., (e.g., affinity affinity matured). Human matured). HumanVL VL and and VH sequences VH sequences from suitable from suitable cells B of B cells of immunized immunized mice asmice as described herein that described herein that express express affinity affinity matured matured antibodies having reverse antibodies having reverse chimeric chimeric heavy heavy chains (i.e., chains (i.e., human human variable variable and mouseconstant) and mouse constant)can canbebe identified and identified andcloned clonedininframe frameininan an expression vector with expression vector with aa suitable suitable human constantregion human constant regiongene gene sequence sequence (e.g., (e.g., a human a human

IgG1). Two IgG1). Twosuch such constructs constructs can can be be prepared, prepared, wherein wherein eacheach construct construct encodes encodes a human a human

heavy chain heavy chain variable variable domain domainthat thatbinds bindsaadifferent different epitope. Oneofofthe epitope. One the human humanVLsVLs (e.g., (e.g.,

humanVk1-39Jk5 human V1-39J5 or human or human VK3-20Ji1), V3-20Jk1), in germline in germline sequence sequence or from or a Bfrom B cell wherein cella wherein the the sequence hasbeen sequence has been somatically somatically mutated, mutated, cancan be fused be fused in frame in frame to atosuitable a suitable human human

constant region constant region gene gene(e.g., (e.g., aa human constantgene). human KKconstant gene).These These three three fully-human fully-human heavy heavy and and light constructs light canbe be constructs can placed placed in a in a suitable suitable cell cell for expression. for expression. The The cell willcell will express express two two major species: major species: aahomodimeric homodimeric heavy heavy chain chain withwith the the identicallight identical lightchain, chain, and andaa heterodimeric heterodimeric heavy heavy chainchain withidentical with the the identical light chain. light chain. To allowTo forallow for aseparation a facile facile separation of of these major these major species, species,one oneofofthe the heavy heavychains chainsisismodified modifiedtoto omit omit aa Protein Protein A-binding A-binding determinant, resulting determinant, resulting in in a differential a differential affinityofofa ahomodimeric affinity homodimeric binding binding proteinprotein from a from a

heterodimeric binding heterodimeric binding protein. protein. Compositions Compositionsandand methods methods thatthat address address thisthis issue issue are are

described in USSN described in USSN12/832,838, 12/832,838, filed2525June filed June 20010, 20010, entitled"Readily entitled IsolatedBispecific "ReadilyIsolated Bispecific Antibodies with Antibodies with Native Native Immunoglobulin ImmunoglobulinFormat," Format," published published as as US US 2010/0331527A1, 2010/0331527A1, hereby hereby

incorporated by reference. incorporated by reference.

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[0095]In

[0095] In one aspect, an one aspect, an epitope-binding epitope-binding protein protein as as described describedherein hereinisis provided, wherein provided, wherein human human VLVL and and VH VH sequences sequences are derived are derived from from mice described mice described herein herein that been that have have been immunized withananantigen immunized with antigencomprising comprising an an epitope epitope of of interest. interest.

[0096]In

[0096] In one embodiment, one embodiment, an an epitope-binding epitope-binding protein protein is isprovided providedthat thatcomprises comprises a first and a first and secondpolypeptide, aa second polypeptide, the first the first polypeptide polypeptide comprising, comprising, from N-terminal from N-terminal to C-terminal, to C-terminal, a first a first epitope-binding epitope-binding region region thatthat selectively selectively bindsbinds a first a first epitope, epitope, followed followed by a constant by a constant region region that comprises that first CH3 comprises aa first region of CH3 region of aa human selectedfrom IgGselected human IgG fromIgG1, IgG1,IgG2, IgG2,IgG4, andand IgG4, a a combination thereof; combination thereof; and, and, aa second secondpolypeptide polypeptidecomprising, comprising,from from N-terminal N-terminal to to C-terminal,a C-terminal, a secondepitope-binding second epitope-bindingregion regionthat that selectively selectively binds a second binds a epitope, followed second epitope, followed by by aa constant region constant region that that comprises secondCH3 comprises aa second CH3 region region of of a human a human IgG IgG selected selected from from IgG1, IgG1,

IgG2, IgG2, IgG4, andaa combination igG4, and combinationthereof, thereof,wherein whereinthe thesecond secondCH3CH3 region region comprises comprises a a modification modification that that reduces or eliminates reduces or eliminates binding binding of ofthe thesecond second CH3 domain CH3 domain to to proteinA.A. protein

[0097]In

[0097] In one embodiment, one embodiment, thesecond the second CH3CH3 region region comprises comprises an modification an H95R H95R modification (by (by IMGT exon numbering; IMGT exon numbering; H435R H435RbybyEU EUnumbering). numbering). InIn another another embodiment, the second embodiment, the second CH3 CH3 region further region further comprises Y96Fmodification comprises aa Y96F modification(IMGT; (IMGT;Y436F Y436F by EU). by EU).

[0098]In

[0098] one embodiment, In one embodiment, the the second second CH3CH3 region region is from is from a modified a modified human human IgG1, IgG1, and and further further comprises modification selected comprises aa modification selected from from the the group group consisting consisting of of D16E, L18M, D16E, L18M, N44S, N44S, K52N, V57M, K52N, V57M, and and V82I V821 (IMGT; (IMGT; D356E, D356E, L358M, L358M,N384S, N384S,K392N, K392N,V397M, V397M, and and V4221bybyEU). V422I EU).

[0099]In

[0099] In one embodiment, one embodiment, thethe second second CH3CH3 region region is from is from a modified a modified human human IgG2, igG2, and and further further comprises modification selected comprises aa modification selected from from the the group group consisting consisting of of N44S, K52N, N44S, K52N, and and

V821 (IMGT;N384S, V82I (IMGT; N384S, K392N, K392N, and and V4221 V422I by EU). by EU).

[00100]

[00100] In one In one embodiment, thesecond embodiment, the secondCH3CH3 region region is from is from a modified a modified human human IgG4, IgG4,

and further and further comprises modification selected comprises aa modification selected from from the the group groupconsisting consistingof of Q15R, N44S, Q15R,N44S, K52N, V57M, K52N, V57M, R69K, R69K, E79Q, E79Q,and andV82I V821 (IMGT; (IMGT; Q355R, Q355R,N384S, N384S,K392N, V397M, K392N,V397M, R409K, R409K,

E419Q,and E419Q, andV422I V4221 by by EU). EU).

[00101]

[00101] One method One method formaking for makingan an epitope-binding epitope-binding protein protein that that binds binds more more than than one one

epitope is to epitope is to immunize first mouse immunize aa first in accordance mouse in with the accordance with the invention invention with an antigen that an antigen that comprises first epitope comprises aafirst epitope of ofinterest, interest,wherein whereinthe mouse the mouse comprises an endogenous comprises an endogenous immunoglobulin light chain immunoglobulin light chain variable variable region region locus locus that that does not contain an endogenous does not endogenous mouse mouse VLVL thatisis capable that capableofofrearranging rearrangingand andforming forminga alight light chain, chain, wherein wherein atat the the endogenous endogenous mouse mouse immunglobulin immunglobulin light light chainchain variable variable region region locus locus is aissingle a single human human VL VL genesegment gene segment operably operably linked linked to to themouse the mouse endogenous endogenous light light chain chain constant constant region region gene,gene,

and the and the human VL gene human VL gene segment segment is is selected selectedfrom human froma a Vk1-39Jk5 and humanV1-39JK5 and aahuman human V3 3-

andthe 20JK1, and 20JK1, theendogenous endogenous mouse mouse VH segments VH gene gene segments have have been been replaced replaced in whole in whole or in or in part with part with human VHgene human VH gene segments, segments, suchsuch thatthat immunoglobulin immunoglobulin heavy heavy chainschains made bymade the by the mouseare mouse aresolely solelyororsubstantially substantially heavy chainsthat heavy chains that comprise comprisehuman human variable variable domains domains and and

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mouseconstant mouse constantdomains. domains. WhenWhen immunized, immunized, such a such mouse mousea will willa reverse make make a chimeric reverse chimeric antibody, antibody, comprising only one comprising only one of of two two human humanlight chainvariable lightchain variabledomains domains(e.g., (e.g.,one oneofof human VK1-39JK5 human Vk1-39Jk5 or human or human V3-20Jk1). Once a BOnce Vx3-20JK1). cell is identified cell aisB identified that encodes that encodes a VH that a VH that

binds theepitope binds the epitopeof of interest, interest, thethe nucleotide nucleotide sequence sequence of (and, of the VH the VH (and, optionally, optionally, the VL) canthe VL) can

be retrieved(e.g., be retrieved (e.g.,bybyPCR) and and PCR) cloned cloned into aninto an expression expression construct construct in frame in frame with with a suitable a suitable

human immunoglobulin human immunoglobulin constant constant domain. domain. This This process process can becan be repeated repeated to identify to identify a second a second

VHdomain VH domainthat thatbinds bindsa asecond second epitope, epitope, and and a second a second VH gene VH gene sequence sequence can be can be retrieved retrieved

and cloned and cloned into into an an expression expressionvector vectorinin frame frame to to aa second secondsuitable suitable immunoglobulin immunoglobulin constant constant

domain. The domain. The first and first and the the second secondimmunoglobulin immunoglobulin constant constant domains domains cansame can the the same or or different isotype, different isotype,and andone one of ofthe theimmunoglobulin constant domains immunoglobulin constant domains(but (butnot notthe theother) other) can canbebe modified as described modified as described herein herein or or in in US 2010/0331527A1, US 2010/0331527A1, and and epitope-binding epitope-binding protein protein can can be be

expressed expressed in in a suitable a suitable cellcell and and isolated isolated basedbased on its on its differential differential affinity affinity for Protein for Protein A as A as

compared compared totoa ahomodimeric homodimeric epitope-binding epitope-binding protein, protein, e.g.,asasdescribed e.g., described in inUSUS

2010/0331527A1. 2010/0331527A1.

[00102]

[00102] In In one one embodiment, method embodiment, a amethod forfor making making a bispecific a bispecific epitope-binding epitope-binding protein protein

is is provided, comprising provided, comprising identifying identifying a first a first affinity-matured affinity-matured (e.g., (e.g., comprising comprising one or one more or more

somatic hypermutations)human somatic hypermutations) humanVH VH nucleotide nucleotide sequence sequence (VH1) (VH1) from afrom mouse mousea as as described described

herein, herein, identifying identifyinga asecond second affinity-matured affinity-matured(e.g., (e.g.,comprising comprisingone oneorormore more somatic somatic

hypermutations)human hypermutations) humanVH VH nucleotide nucleotide sequence sequence (VH2) (VH2) from afrom a mouse mouse as described as described herein, herein, cloning cloning VH1 in frame VH1 in frame with with aa human humanheavy heavy chain chain lacking lacking a Protein a Protein A-determinant A-determinant modification modification

as described in as described in US 2010/0331527A1 US 2010/0331527A1 for for form form heavy heavy chain chain 1 (HC1), 1 (HC1), cloning cloning VH2 VH2 in in frame frame

with aa human with heavy human heavy chain chain comprising comprising a Protein a Protein A-determinant A-determinant as described as described in USin US 2010/0331527A1 2010/0331527A1 to to form form heavy heavy chain chain 2 (HC2), 2 (HC2), introducing introducing an expression an expression vector vector comprising comprising

HC1 andthe HC1and thesame sameor or a differentexpression a different expressionvector vectorcomprising comprising HC2HC2 intointo a cell,wherein a cell, wherein the the

cell cell also alsoexpresses a human expresses a immunoglobulin human immunoglobulin light light chain chain thatcomprises that comprises a human a human 1- V1

39/human J5ororaa human 39/human J5 humanV3-20/human Vx3-20/human JK1 fused J1 fused to ato human a human lightchain light chainconstant constant domain, allowing domain, allowing the the cell cell to toexpress express aa bispecific bispecificepitope-binding epitope-bindingprotein proteincomprising comprising aa VH VH

domainencoded domain encodedby by VH1VH1 and and VH domain a VHadomain encoded encoded by VH2, by andVH2, and isolating isolating the bispecific the bispecific

epitope-binding epitope-binding protein protein based based ondifferential on its its differential ability ability to bind to bind Protein Protein A as compared A as compared with a with a monospecific homodimeric monospecific homodimeric epitope-binding epitope-binding protein. protein. In In a specificembodiment, a specific embodiment, HC1 HC1 is anis an

IgG1, and IgG1, and HC2 HC2isisananIgG1 IgG1that thatcomprises comprisesthethe modificationH95R modification H95R H435RH435R (MGT; (IMGT; by EU)by EU) and and further comprises further the modification comprises the modification Y96F (IMGT;Y436F Y96F (IMGT; Y436F by EU). by EU). In one In one embodiment, embodiment, the VHthe VH domainencoded domain encodedby by VH1, VH1, the the VH domain VH domain encoded encoded by VH2,byorVH2, both,orare both, are somatically somatically mutated. mutated.

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HumanVH Human VHGenes Genes That That Express Express with witha aCommon HumanVL Common Human VL

[00103]

[00103] A variety A variety of of human variable regions human variable regions from from affinity-matured affinity-matured antibodies antibodies raised raised againstfour against fourdifferent differentantigens antigens were were expressed expressed with their with either eithercognate their cognate light light chain, or chain, at or at least one least of aa human one of light chain human light chain selected selected from from human VK1-39JK5, humanVk1-39JK5, human human VK3-20JK1, V3-20Jk1, or or humanVpreBJX5 human VpreBJk5 (see(see Example Example 1). antibodies 1). For For antibodies to each to each of antigens, of the the antigens, somatically somatically

mutatedhigh mutated high affinityheavy affinity heavy chains chains from different from different gene families gene families paired successfully paired successfully with with rearranged human rearranged human germline germline VK1-39JK5 Vk1-39Jk5 and VK3-20J1 and V3-20Jk1 regions regions and wereand were secreted secreted from from cells expressing cells expressing the the heavy andlight heavy and light chains. chains. For V1-39J5andand For Vk1-39Jk5 V3-20JK1, 3-20J1, VH VH domains domains derived from derived from the the following following human human VHVH familiesexpressed families expressed favorably: favorably: 1-2,1-8, 1-2, 1-8,1-24, 1-24,2-5, 2-5, 3-7, 3-7, 3-9, 3-11, 3-9, 3-11,3-13, 3-13,3-15, 3-15,3-20, 3-20, 3-23, 3-23, 3-30, 3-30, 3-33,3-33, 3-48,3-48, 4-31, 4-31, 4-39,5-51, 4-39, 4-59, 4-59,and 5-51, 6-1. and Thus,6-1. a Thus, a mousethat mouse thatisis engineered engineeredtoto express expressa alimited repertoire of limited repertoire of human VLdomains human VL domains from from one one or or both of both of VK1-39JK5 andVk3-20Jk1 Vk1-39Jk5 and VK3-20J1willwill generate generate a diverse a diverse population population of somatically of somatically mutated mutated

human human VHVH domains domains fromfrom VH locus a VHa locus modified modified to replace to replace mousemouse VHsegments VH gene gene segments with with human VH human VHgene genesegments. segments.

[00104]

[00104] Mice genetically Mice genetically engineered to express engineered to expressreverse reversechimeric chimeric(human (human variable, variable,

mouseconstant) mouse constant)immunoglobulin immunoglobulin heavy heavy chains chains associated associated with with a single a single rearranged rearranged lightlight

chain (e.g., chain (e.g., aaVK1-39/J Vk1-39/J or or aa VK3-20/J), Vk3-20/J), when immunizedwith when immunized withanan antigen antigen of of interest, interest,

generated B Bcells generated cells that that comprised diversity of comprised aadiversity of human segment human V Vsegment rearrangements rearrangements and and expressed expressed a diversity a diversity of high-affinity of high-affinity antigen-specific antigen-specific antibodies antibodies with diverse with diverse properties properties with with respecttototheir respect theirability ability to to block blockbinding bindingof of thethe antigen antigen to its to its ligand, ligand, and and with with respect respect to to their their ability to ability to bind bind variants of the variants of theantigen antigen (see (see Examples Examples 5 through 5 through 10). 10).

[00105]

[00105] Thus, the Thus, the mice mice and andmethods methods described described herein herein are are useful useful in in making making and and

selecting human selecting immunoglobulin human immunoglobulin heavy heavy chain chain variable variable domains, domains, including including somatically somatically

mutated human mutated human heavy heavy chain chain variable variable domains, domains, thatthat result result fromfrom a diversityofof a diversity

rearrangements, rearrangements, thatthat exhibit exhibit a wide a wide variety variety of affinities of affinities (including (including exhibiting exhibiting KD ofa a KD ofaabout about a nanomolar nanomolar or less), or less), a wide a wide variety variety of specificities of specificities (including (including binding binding to different to different epitopes epitopes of of the the same antigen), and same antigen), andthat that associate associateand andexpress expresswith withthe thesame sameor or substantiallythe substantially thesame same humanimmunoglobulin human immunoglobulin light light chain chain variableregion. variable region.

[00106]

[00106] The following The following examples examplesare areprovided providedsoso asas totodescribe describetotothose thoseofofordinary ordinary skill inin skill thethe artart howhow to to make makeand and use use methods andcompositions methods and compositionsof of theinvention, the invention,and andare arenot not intendedtotolimit intended limitthe thescope scope of what of what the inventors the inventors regardregard asinvention. as their their invention. Efforts Efforts have been have been madetotoensure made ensureaccuracy accuracy with with respect respect to to numbers numbers usedused (e.g., (e.g., amounts, amounts, temperature, temperature, etc.)etc.)

but some but experimentalerrors some experimental errorsand anddeviations deviationsshould should be be accounted accounted for.for. Unless Unless indicated indicated

otherwise, parts otherwise, parts are are parts parts by by weight, weight, molecular weight is molecular weight is average molecularweight, average molecular weight, temperature temperature is indicated is indicated in Celsius, in Celsius, and pressure and pressure is at orisnear at or near atmospheric. atmospheric.

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EXAMPLES EXAMPLES

Example Example 1.1.Identification Identification ofof human human heavy heavy chainchain variable variable regions regions that associate that associate with with May selected human selected human light light chain chain variable variable regions regions

[00107]

[00107] An in An in vitro vitro expression expression system wasconstructed system was constructedtotodetermine determineififaa single single rearranged human rearranged human germline germline lightchain light chaincould couldbebeco-expressed co-expressed withwith human human heavyheavy chainschains

from antigen from antigen specific specific human antibodies. human antibodies.

[00108]

[00108] Methodsfor Methods for generating generatinghuman human antibodies antibodies in in geneticallymodified genetically modifiedmice mice areare

known (see e.g., known (see e.g.,USUS6,596,541, 6,596,541,Regeneron RegeneronPharmaceuticals, Pharmaceuticals,VELOCIMMUNE@). The VELOCIMMUNE®). The VELOCIMMUNE@ VELOCIMMUNE® technology technology involves involves generation generation of a genetically of a genetically modified modified mouseahaving mouse having a genomecomprising genome comprising human human heavy heavy and light and light chainchain variable variable regions regions operably operably linked linked to to endogenous mouse endogenous mouse constant constant region region loci loci suchsuch thatthat thethe mouse mouse produces produces an antibody an antibody

comprising humanvariable comprising aa human variableregion regionandand a mouse a mouse constant constant region region in response in response to antigenic to antigenic

stimulation. The stimulation. DNAencoding The DNA encoding thethe variable variable regions regions of of theheavy the heavy andand lightchains light chainsofofthe the antibodies producedfrom antibodies produced froma aVELOCIMMUNE® VELOCIMMUNE@ mouse mouse are fully are fully Initially, human. human. Initially, high affinity high affinity

chimeric antibodies are chimeric antibodies are isolated isolated having a human having a variableregion human variable regionand anda amouse mouse constant constant

region. As described region. As describedbelow, below,the theantibodies antibodiesare arecharacterized characterizedand andselected selectedforfordesirable desirable characteristics, includingaffinity, characteristics, including affinity,selectivity, selectivity,epitope, epitope,etc. etc.TheThe mouse mouse constant constant regions are regions are

replaced with replaced with aa desired humanconstant desired human constant regiontotogenerate region generate human a fully human a fully antibody antibody

containing non-IgMisotype, a non-IgM containing a isotype, for for example, wild-type or example, wild-type or modified modified IgG1, IgG1, IgG2, IgG3ororIgG4. IgG2, IgG3 IgG4. While theconstant While the constant region region selected selected mayaccording may vary vary according touse, to specific specific use, high high affinity affinity antigen antigen-

bindingand binding andtarget target specificity specificity characteristics characteristics reside reside in variable in the the variable region. region.

[00109]

[00109] A A VELOCIMMUNE@ VELOCIMMUNE® mousemouse was immunized was immunized with awith a growth growth factor factor that that promotesangiogenesis promotes angiogenesis (Antigen (Antigen C) C) andand antigen-specific antigen-specific human human antibodies antibodies werewere isolated isolated

and sequenced and sequenced forforV V gene gene usage usage using using standard standard techniques techniques recognized recognized in theinart. the art. Selected Selected

antibodies were cloned antibodies were clonedonto ontohuman human heavy heavy and and light light chain chain constant constant regions regions and and 69 heavy 69 heavy

chains were chains were selected selected for for pairing pairing with with one one of of three three human human light light (1) chains: chains: (1) theK light the cognate cognateK light chain linked to chain linked to aa human constantregion, human Ki constant region, (2) (2) aa rearranged human rearranged human germline germline VK1-39JK5 Vk1-39Jk5

linked linked to to aahuman constantregion, human Ki constant region, or or (3) (3) aa rearranged humangermline rearranged human germline Vx3-20JK 3-20J1 linked linked

to aa human to constantregion. human Ki constant region.Each Each heavy heavy chain chain and and light light chain chain pair pair were were co-transfected co-transfected in in CHO-K1 cellsusing CHO-K1 cells usingstandard standard techniques. techniques. Presence Presence of antibody of antibody in the in the supernatant supernatant was was

detected by detected by anti-human anti-humanIgG IgGininananELISA ELISA assay. assay. Antibody Antibody titer titer (ng/ml) (ng/ml) waswas determined determined for for eachheavy each heavy chain/light chain/light chain chain pair pair and titers and titers withdifferent with the the different rearranged rearranged germline germline light light chains were chains werecompared comparedto to thetiters the titers obtained obtainedwith with the the parental parental antibody antibody molecule molecule(i.e., (i.e., heavy heavy

chainpaired chain pairedwith with cognate cognate lightlight chain) chain) and percent and percent of titer of native nativewastiter was calculated calculated (Table 1).(Table 1).

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variablegene. chainvariable Heavychain no no ND: gene.ND: expression detected under current experimental 2023202781 04 May 2023

VH: VH: Heavy expression detected under current experimental

conditions. conditions.

[00110]

[00110]

Table 11 Table

Antibody (ng/ml) Titer(ng/ml) Antibody Titer Percent ofNative Percent of NativeTiter Titer VH Cognate LC Cognate LC Vicl-39Jic5 VK1-39JK5 Vi3-2OJcl V3-20Jk1 Vicl-39Jc5 Vk1-39JK5 Vic3-20JK1 VK3-20JK1 3-15 3-15 63 63 23 23 11 11 36.2 36.2 17.5 17.5

1-2 1-2 103 103 53 53 ND ND 51.1 51.1 -

3-23 3-23 83 83 60 60 23 23 72.0 72.0 27.5 27.5

3-33 3-33 15 15 77 77 ND ND 499.4 499.4 -

4-31 4-31 22 22 69 69 17 17 309.4 309.4 76.7 76.7

3-7 3-7 53 53 35 35 28 28 65.2 65.2 53.1 53.1

- - 22 22 32 32 19 19 148.8 148.8 89.3 89.3

1-24 1-24 3 3 13 13 ND ND 455.2 455.2 -

3-33 3-33 11 47 47 ND ND 5266.7 5266.7 -

3-33 3-33 58 58 37 37 ND ND 63.1 63.1 -

-- 110 110 67 67 18 18 60.6 60.6 16.5 16.5

3-23 3-23 127 127 123 123 21 21 96.5 96.5 16.3 16.3

3-33 3-33 28 28 16 16 2 2 57.7 57.7 7.1 7.1

3-23 3-23 32 32 50 50 38 38 157.1 157.1 119.4 119.4 - - 18 18 45 45 18 18 254.3 254.3 101.7 101.7

3-9 3-9 1 1 30 30 23 23 2508.3 2508.3 1900.0 1900.0

3-11 3-11 12 12 26 26 66 225.9 225.9 48.3 48.3

1-8 1-8 16 16 ND ND 13 13 -- 81.8 81.8

3-33 3-33 54 54 81 81 10 10 150.7 150.7 19.1 19.1

- 34 34 9 9 ND 25.9 25.9 - ND -

3-20 3-20 7 7 14 14 54 54 203.0 203.0 809.0 809.0 3-33 3-33 19 19 38 38 ND ND 200.5 200.5 I

3-11 3-11 48 48 ND ND 203 203 -- 423.6 423.6 - - 11 11 23 23 8 8 212.7 212.7 74.5 74.5

3-33 3-33 168 168 138 138 182 182 82.0 82.0 108.2 108.2

3-20 3-20 117 117 67 67 100 100 57.5 57.5 86.1 86.1

3-23 3-23 86 86 61 61 132 132 70.7 70.7 154.1 154.1

3-33 3-33 20 20 12 12 33 33 60.9 60.9 165.3 165.3

4-31 4-31 69 69 92 92 52 52 133.8 133.8 75.0 75.0

3-23 3-23 87 87 78 78 62 62 89.5 89.5 71.2 71.2

1-2 1-2 31 31 82 82 51 51 263.0 263.0 164.6 164.6

3-23 3-23 53 53 93 93 151 151 175.4 175.4 285.4 285.4

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04 May 2023

- - 11 11 88 17 17 75.7 75.7 151.4 151.4

3-33 3-33 114 114 36 36 27 27 31.6 31.6 23.4 23.4

3-15 3-15 73 73 39 39 44 44 53.7 53.7 59.6 59.6

3-33 3-33 1 1 34 34 16 16 5600.0 5600.0 2683.3 2683.3 3-9 3-9 58 58 112 112 57 57 192.9 192.9 97.6 97.6

3-33 3-33 67 67 20 20 105 105 30.1 30.1 157.0 157.0

3-33 3-33 34 34 21 21 24 24 62.7 62.7 70.4 70.4

2023202781 3-20 3-20 10 10 49 49 91 91 478.4 478.4 888.2 888.2 3-33 3-33 66 66 32 32 25 25 48.6 48.6 38.2 38.2

3-23 3-23 17 17 59 59 56 56 342.7 342.7 329.8 329.8 - - 58 58 108 108 19 19 184.4 184.4 32.9 32.9

-- 68 68 54 54 20 20 79.4 79.4 29.9 29.9

3-33 3-33 42 42 35 35 32 32 83.3 83.3 75.4 75.4

-- 29 29 19 19 13 13 67.1 67.1 43.9 43.9

3-9 3-9 24 24 34 34 29 29 137.3 137.3 118.4 118.4

3-30/33 3-30/33 17 17 33 33 7 7 195.2 195.2 43.1 43.1

3-7 3-7 25 25 70 70 74 74 284.6 284.6 301.6 301.6

3-33 3-33 87 87 127 127 ND ND 145.1 145.1 -

6-1 6-1 28 28 56 56 ND ND 201.8 201.8 -

3-33 3-33 56 56 39 39 20 20 69.9 69.9 36.1 36.1

3-33 3-33 10 10 53 53 1 1 520.6 520.6 6.9 6.9

3-33 3-33 20 20 67 67 10 10 337.2 337.2 52.3 52.3

3-33 3-33 11 11 36 36 18 18 316.8 316.8 158.4 158.4

3-23 3-23 12 12 42 42 32 32 356.8 356.8 272.9 272.9 3-33 3-33 66 66 95 95 15 15 143.6 143.6 22.5 22.5

3-15 3-15 55 55 68 68 ND ND 123.1 123.1 -

- - 32 32 68 68 3 3 210.9 210.9 10.6 10.6

1-8 1-8 28 28 48 48 ND ND 170.9 170.9 -

3-33 3-33 124 124 192 192 21 21 154.3 154.3 17.0 17.0

3-33 3-33 0 0 113 113 ND ND 56550.0 56550.0 -

3-33 3-33 I 10 10 157 157 11 1505.8 1505.8 12.5 12.5

3-33 3-33 6 6 86 86 15 15 1385.5 1385.5 243.5 243.5 3-23 3-23 70 70 115 115 22 22 163.5 163.5 31.0 31.0

3-7 3-7 71 71 117 117 21 21 164.6 164.6 29.6 29.6

3-33 3-33 82 82 100 100 47 47 122.7 122.7 57.1 57.1

3-7 3-7 124 124 161 161 41 41 130.0 130.0 33.5 33.5

[00111]

[00111] In aa similar In similarexperiment, experiment,VELOCIMMUNE@ miceimmunized VELOCIMMUNE® mice were were immunized with with several several different antigens different antigens and and selected selected heavy chainsofof antigen heavy chains antigen specific specific human antibodieswere human antibodies were testedfor tested fortheir their ability ability to to pair pair with different rearranged with different rearranged human human germline germline light (as light chains chains (as

27

WO2011/097603 wo 2011/097603 PCT/US2011/023971 PCT/US2011/023971

described above). above). The The antigens used in in thisexperiment experiment included an an enzyme involved in 2023202781 04 May 2023

described antigens used this included enzyme involved in

cholesterol homeostasis cholesterol (AntigenA), homeostasis (Antigen A), aa serum serumhormone hormone involved involved in regulating in regulating glucose glucose

homeostasis(Antigen homeostasis (AntigenB), B),aagrowth growthfactor factorthat that promotes promotesangiogenesis angiogenesis (Antigen (Antigen C) C) andand a cell a cell-

surface receptor surface receptor (Antigen (Antigen D). D). Antigen Antigen specific specific antibodies antibodies were wereisolated isolated from from mice miceofof each each immunizationgroup immunization groupand andthe theheavy heavy chain chain andand lightchain light chainvariable variableregions regionswere were cloned cloned andand

sequenced.From sequenced. From thethe sequence sequence of the of the heavy heavy and light and light chains, chains, V gene V gene usageusage was was determinedand determined andselected selectedheavy heavy chains chains were were paired paired withwith either either theircognate their cognate lightchain light chainoror aa rearranged human rearranged human germline germline VK1-39J5 Vk1-39Jk5 region. region. Each Each heavy/light heavy/light chain chain pairco- pair was was co transfected in CHO-K1 transfected in cellsand CHO-K1 cells andthe thepresence presenceofof antibody antibody in inthe thesupernatant supernatantwaswas detected detected

by anti-human by anti-humanIgG igGinin an anELISA ELISA assay. assay. Antibody Antibody titer titer (pg/ml)was (µg/ml) was determined determined for for each each heavy heavy

chain/light chain chain/light chainpairing pairingandand titers titers with with thethe different different rearranged rearranged human human germline germline light light chains chains were compared were compared to to thetiters the titers obtained obtained with with the the parental parental antibody antibody molecule molecule(i.e., (i.e., heavy chain heavy chain

paired with paired withcognate cognate light light chain) chain) and and percent percent of native of native titercalculated titer was was calculated (Table (Table 2). VH: 2). VH

Heavychain Heavy chainvariable variablegene. gene.: Vi: K light K light chain chain variable variable gene. gene. ND: ND: no expression no expression detected detected

under current under current experimental experimental conditions. conditions.

[00112]

[00112]

_ _ Table22 Table

Titer (pgrnl) Titer (µg/ml) Percentof Percent of Antigen Antigen Antibody Antibody VH VH V A e VH + VH + NativeTiter Alone VH VH Alone VH VH ++VK Vi1-39Jr5 Native Titer VK1-39JK5 320 320 1-18 1-18 2-30 2-30 0.3 0.3 3.1 3.1 2.0 2.0 66 66 321 321 2-5 2-5 2-28 2-28 0.4 0.4 0.4 0.4 1.9 1.9 448 448 334 334 2-5 2-5 2-28 2-28 0.4 0.4 2.7 2.7 2.0 2.0 73 73 A A 313 313 3-13 3-13 3-15 3-15 0.5 0.5 0.7 0.7 4.5 4.5 670 670 316 316 3-23 3-23 4-1 4-1 0.3 0.3 0.2 0.2 4.1 4.1 2174 2174 315 315 3-30 3-30 4-1 4-1 0.3 0.3 0.2 0.2 3.2 3.2 1327 1327 318 318 4-59 4-59 1-17 1-17 0.3 0.3 4.6 4.6 4.0 4.0 86 86 257 257 3-13 3-13 1-5 1-5 0.4 0.4 3.1 3.1 3.2 3.2 104 104 283 283 3-13 3-13 1-5 1-5 0.4 0.4 5.4 5.4 3.7 3.7 69 69 637 637 3-13 3-13 1-5 1-5 0.4 0.4 4.3 4.3 3.0 3.0 70 70 638 638 3-13 3-13 1-5 1-5 0.4 0.4 4.1 4.1 3.3 3.3 82 82 B B 624 624 3-23 3-23 1-17 1-17 0.3 0.3 5.0 5.0 3.9 3.9 79 79 284 284 3-30 3-30 1-17 1-17 0.3 0.3 4.6 4.6 3.4 3.4 75 75 653 653 3-33 3-33 1-17 1-17 0.3 0.3 4.3 4.3 0.3 0.3 77 268 268 4-34 4-34 1-27 1-27 0.3 0.3 5.5 5.5 3.8 3.8 69 69 633 633 4-34 4-34 1-27 1-27 0.6 0.6 6.9 6.9 3.0 3.0 44 44 C C 730 730 3-7 3-7 1-5 1-5 0.3 0.3 1.1 1.1 2.8 2.8 249 249 728 728 3-7 3-7 1-5 1-5 0.3 0.3 2.0 2.0 3.2 3.2 157 157

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691 3-9 3-20 0.3 2023202781 04 May 2023

691 3-9 3-20 0.3 2.8 2.8 3.1 3.1 109 109 749 749 3-33 3-33 3-15 3-15 0.3 0.3 3.8 3.8 2.3 2.3 62 62 750 750 3-33 3-33 1-16 1-16 0.3 0.3 3.0 3.0 2.8 2.8 92 92 724 724 3-33 3-33 1-17 1-17 0.3 0.3 2.3 2.3 3.4 3.4 151 151 706 706 3-33 3-33 1-16 1-16 0.3 0.3 3.6 3.6 3.0 3.0 84 84 744 744 1-18 1-18 1-12 1-12 0.4 0.4 5.1 5.1 3.0 3.0 59 59 696 696 3-11 3-11 1-16 1-16 0.4 0.4 3.0 3.0 2.9 2.9 97 97 685 685 3-13 3-13 3-20 3-20 0.3 0.3 0.5 0.5 3.4 3.4 734 734 732 732 3-15 3-15 1-17 1-17 0.3 0.3 4.5 4.5 3.2 3.2 72 72 694 694 3-15 3-15 1-5 1-5 0.4 0.4 5.2 5.2 2.9 2.9 55 55 743 743 3-23 3-23 1-12 1-12 0.3 0.3 3.2 3.2 0.3 0.3 10 10 742 742 3-23 3-23 2-28 2-28 0.4 0.4 4.2 4.2 3.1 3.1 74 74 693 693 3-23 3-23 1-12 1-12 0.5 0.5 4.2 4.2 4.0 4.0 94 94 136 136 3-23 3-23 2-28 2-28 0.4 0.4 5.0 5.0 2.7 2.7 55 55 155 155 3-30 3-30 1-16 1-16 0.4 0.4 1.0 1.0 2.2 2.2 221 221 163 163 3-30 3-30 1-16 1-16 0.3 0.3 0.6 0.6 3.0 3.0 506 506 171 171 3-30 3-30 1-16 1-16 0.3 0.3 1.0 1.0 2.8 2.8 295 295 145 145 3-43 3-43 1-5 1-5 0.4 0.4 4.4 4.4 2.9 2.9 65 65 49 3-48 3-48 3-11 3-11 0.3 0.3 1.7 1.7 2.6 2.6 155 155 D D 49 51 51 3-48 3-48 1-39 1-39 0.1 0.1 1.9 1.9 0.1 0.1 4 4 159 159 3-7 3-7 6-21 6-21 0.4 0.4 3.9 3.9 3.6 3.6 92 92 169 169 3-7 3-7 6-21 6-21 0.3 0.3 1.3 1.3 3.1 3.1 235 235 134 134 3-9 3-9 1-5 1-5 0.4 0.4 5.0 5.0 2.9 2.9 58 58 141 141 4-31 4-31 1-33 1-33 2.4 2.4 4.2 4.2 2.6 2.6 63 63 142 142 4-31 4-31 1-33 1-33 0.4 0.4 4.2 4.2 2.8 2.8 67 67

[00113]

[00113] The results The results obtained from these obtained from these experiments experimentsdemonstrate demonstrate thatthat somatically somatically

mutated,high mutated, high affinityheavy affinity heavy chains chains from from different different gene families gene families are ableare ablewith to pair to pair with rearranged human rearranged human germline germline VK1-39JK5 Vk1-39Jk5 and VK3-20JKI and Vk3-20Jk1 regionsregions and be and be secreted secreted from thefrom the

cell cell as as aa normal normal antibody molecule. AsAsshown antibody molecule. shownin in Table Table 1, 1, antibody antibody titer was titer wasincreased increasedfor for about 61%(42 about 61% (42ofof69) 69)heavy heavychains chains when when paired paired with with thethe rearranged rearranged human human Vk1-39Jk5 light light V1-39JK5 chain and about chain and about29% 29%(20(20ofof69) 69)heavy heavy chains chains when when paired paired withwith the the rearranged rearranged human human 3- V3 20J1light 20Jk1 light chain chain as as compared compared totothe thecognate cognatelight light chain chain of of the the parental parental antibody. For about antibody. For about 20%(14 20% (14ofof69) 69) of of the the heavy heavychains, chains, both rearrangedhuman both rearranged human germline germline light light chains chains conferred conferred

an increase an increase in in expression as compared expression as comparedto to thecognate the cognate lightchain light chainofofthe theparental parental antibody. antibody. As shown As shownininTable Table2,2,the the rearranged rearrangedhuman human germline germline regionregion V1-39JK5 Vk1-39Jk5 conferred conferred an an increase increase ininexpression expression of several of several heavyheavy chains chains specificspecific for of for a range a range of different different classes ofclasses of

antigensasas antigens compared compared to thetocognate the cognate lightfor light chain chain for the parental the parental antibodies. antibodies. Antibody Antibody titer titer wasincreased was increasedbybymore more than than two-foldforforabout two-fold about35% 35% (15/43) (15/43) of of thethe heavy heavy chains chains as as

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compared compared totothe thecognate cognatelight lightchain chainof of the the parental parental antibodies. antibodies. For 2023202781 04 May 2023

For two two heavy heavychains chains(315 (315 and 316), the and 316), the increase increase was wasgreater greaterthan thanten-fold ten-fold as as compared comparedto tothe theparental antibody. parentalantibody. Within all Within all the theheavy heavy chains chains that that showed increaseexpression showed increase expressionrelative relative to to the the cognate cognatelight light 3 chain of the chain of the parental parental antibody, antibody, family familythree three(VH ) heavy (VH3) heavy chains chains are are over over represented in represented in

comparison comparison totoother otherheavy heavychain chainvariable variableregion regiongene genefamilies. families.This Thisdemonstrates demonstrates a a

favorable relationship favorable relationship of of human VH 3heavy human VH3 heavychains chains totopair pairwith withrearranged rearrangedhuman human germline germline

VK1-39JK5and Vk1-39Jk5 VK3-20JK1 andVk3-20Jk1 light light chains. chains.

Example 2. Example 2. Generation Generationof of aa Rearranged Rearranged Human Human Germline Germline Light Light Chain Chain Locus Locus

[00114]

[00114] Various rearrangedhuman Various rearranged human germline germline light light chain chain targetingvectors targeting vectors were were made made

using VELOCIGENE@ using VELOCIGENE® technology technology (see, (see, e.g., e.g., US No. US Pat. Pat.6,586,251 No. 6,586,251 and Valenzuela and Valenzuela et al. et al.

(2003) High-throughput (2003) High-throughputengineering engineeringofofthe themouse mouse genome genome coupled coupled with high-resolution with high-resolution

expression analysis, expression analysis, Nature Nature Biotech. Biotech. 21(6):652-659) 21(6):652-659)totomodify modifymouse mouse genomic genomic Bacterial Bacterial

Artificial Chromosome Artificial (BAC)clones Chromosome (BAC) clones 302g12 302g12 and and 254m04 254m04 (Invitrogen). (Invitrogen). Using Using these these two two BAC clones,genomic BAC clones, genomic constructs constructs were were engineered engineered to contain to contain a single a single rearranged rearranged human human

germline light germline light chain chain region region and and inserted inserted into intoan anendogenous light chain endogenous K Klight locus that chain locus that was was

previously modified previously to delete modified to delete the the endogenous variableand endogenous K Kvariable andjoining joininggene genesegments. segments.

A. Construction A. Construction of of Rearranged Rearranged Human Human Germline Germline Light Light Chain Chain Targeting Targeting Vectors Vectors

[00115]

[00115] Three different rearranged Three different humangermline rearranged human germline lightchain light chainregions regionswere were made made

using standard using standard molecular molecularbiology biologytechniques techniquesrecognized recognized in in theart. the art. The Thehuman human variable variable

genesegments gene segments used used forfor constructing constructing these these three three regions regions included included rearranged rearranged human human 1- VK1 39JK5 sequence, 39Jk5 sequence, aa rearranged rearranged human human Vk3-20Jk1 sequenceand VK3-20JK1 sequence andaa rearranged rearranged human human

VpreBJk5 sequence. VpreBJX5 sequence.

[00116]

[00116] A DNA A DNAsegment segment containing containing exonexon 1 (encoding 1 (encoding the leader the leader peptide) peptide) and intron and intron 1 1 of the of themouse mouse VK3-7 gene was V3-7 gene was made madebybydedenovo novoDNA DNAsynthesis synthesis(Integrated (Integrated DNA DNA

Technologies). Technologies). PartPart of the of the 5' untranslated 5' untranslated region region up to a up to a naturally naturally occurringoccurring Bp restriction Bipl restriction

enzyme site was enzyme site was included. included.Exons Exons ofofhuman Vk1-39 and human VKl-39 and V<3-20 geneswere 3-20 genes werePCR PCR amplified amplified

from human from humangenomic genomic BAC BAC libraries. libraries. The The forward forward primers primers had ahad a 5' extension 5' extension containing containing the the splice splice acceptor site ofofintron acceptor site intronI 1 of of thethe mouse mouseVx3-7 gene. The V3-7 gene. The reverse reverse primer primer used used forfor PCRPCR

of of the the human human Vk1-39 sequence VK1-39sequence included included an extension an extension encoding encoding human human JK5, whereas Jk5, whereas the the reverse primer used reverse primer usedfor for PCR PCRofofthe thehuman human V3-20 3-20 sequence sequence includedincluded an extension an extension

encoding human encoding human J1. Thehuman JK1.The human VpreBJ5 VpreBJX5 sequence sequence was was mademade by debynovo de novo DNA DNA synthesis (Integrated synthesis (Integrated DNA DNATechnologies). Technologies).A portion A portion of of thehuman the human J-CK JK-CK intron intron including including thethe

splice donor splice donor site site was was PCR amplifiedfrom PCRamplified fromplasmid plasmidpBS-296-HA18-PIScel. pBS-296-HA18-PIScel. The forward The forward PCR PCR

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primer included included an an extension extension encoding encodingpart partofofeither either aa human humanJ5, JK5, J1,Ji1, or JA5 sequence. 2023202781 04 2023

primer or JX5 sequence.

The reverse The reverseprimer primerincluded includeda aPI-Scel PI-Scelsite, site, which waspreviously which was previouslyengineered engineeredinto intothe theintron. intron.

[00117]

[00117] The mouse The mouse3-7VK3-7 exonl/intron exon1/intron 1, human 1, human variable variable light light chainchain exons, exons, and and May humanJK-CK human intronfragments JK-Cintron fragments were were sewn sewn together together by overlap by overlap extension extension PCR, PCR, digested digested with with Blpl and Blpl PI-Scel, and and PI-Scel, ligated into and ligated intoplasmid plasmid pBS-296-HA18-PiScel, which pBS-296-HA18-PIScel, which contained contained the the

promoter from promoter from the the human human VK3-15 3-15 variable variable gene gene segment. segment. loxed hygromycin A loxed Ahygromycin cassette cassette within plasmid within pBS-296-HA18-PIScel plasmid pBS-296-HA18-PIScel was was replaced replaced with with a FRTed a FRTed hygromycin hygromycin cassettecassette

flanked by Notl flanked by Notl and AscI sites. and Ascl sites. The The Notl/PI-Scel Not/PI-Scel fragment fragmentofofthis this plasmid plasmid was wasligated ligated into into modified mouse modified mouseBAC BAC 254m04, 254m04, whichwhich contained contained part part of theofmouse the mouse intron,intron, JK-CK J-CK the mouse the mouse

exon, and CG exon, CK andabout about7575kbkbofofgenomic genomic sequence sequence downstream downstream of the of the mouse mouse locus K locusKwhich which provided aa 3' provided 3' homology armfor homology arm forhomologous homologous recombination recombination in mouse in mouse ES cells. ES cells. The The Notl/AscIfragment Notl/Ascl of this fragment of this BAC wasthen BAC was thenligated ligated into into modified modified mouse mouseBAC BAC 302g12, 302g12, which which

contained aa FRTed contained FRTedneomycin neomycin cassette cassette and and about about 23 kb23ofkbgenomic of genomic sequence sequence upstream upstream of of the the endogenous endogenous K K locus locus forhomologous for homologous recombination recombination in mouse in mouse ES cells. ES cells.

B. Rearranged B. Rearranged Human Human Germline Germline Vicl-39Jic5 VK1-39JK5 Targeting Targeting Vector Vector (Figure (Figure 1) 1)

[00118]

[00118] Restriction Restriction enzyme sites were enzyme sites wereintroduced introducedatatthe the 5' 5' and and 3' 3' ends of an ends of an engineered engineered light light chain chain insert insert for for cloning cloning into into a targeting a targeting vector: vector: ansite an Ascl AscIat site at the the 5' 5' end end and and a PI-Scelsite a PI-Scel siteatatthe the3'3'end. end.Within the the Within 5'Asc 5' Ascl site site and3'the and the 3' PI-Scel PI-Scel site site the the targeting targeting

construct from construct 5' to from 5' to 3'3'included includeda a5'5' homology homology arm arm containing sequence5'5'toto the containing sequence the endogenous endogenous mouse mouse K light K light chain chain locus locus obtained obtained from from mouse mouse BAC clone BAC clone 302g12, 302g12, a FRTeda FRTed neomycinresistance neomycin resistancegene, gene,anangenomic genomic sequence sequence including including the human the human Vk3-15V3-15 promoter, promoter, a a leader sequence leader sequence ofofthe the mouse mouse3-7Vi3-7 variable variable gene gene segment, segment, a intron a intron sequence sequence of the of the mouseV3-7 mouse VK3-7 variable variable gene gene segment, segment, an open an open reading reading frame frame of a rearranged of a rearranged human human germline germline VK1-39JK5 region,a agenomic VK1-39Ji5region, genomic sequence sequence containing containing a portion a portion of the of the human human JK-CKJ<-CG

intron, and intron, and aa 3'3'homology armcontaining homology arm containingsequence sequence3' 3' ofofthe theendogenous endogenous mouse mouse JK5 Jk5 gene gene segment obtainedfrom segment obtained frommouse mouse BAC BAC cloneclone 254m04 254m04 (Figure(Figure 1, middle). 1, middle). Genes and/or Genes and/or

sequences upstream sequences upstream of of thethe endogenous endogenous mousemouse K light K light chainchain locuslocus and downstream and downstream of the of the

most 3' most 3' JJKgene genesegment segment (e.g.,thetheendogenous (e.g., endogenous 3' enhancer) 3' enhancer) were were unmodified unmodified by theby the targeting construct targeting construct (see (see Figure Figure 1). 1). The sequenceofofthe The sequence theengineered engineered human human VK1-39JK5 Vk1-39JK5

locus is locus is shown in SEQ shown in SEQ IDIDNO:1. NO:1.

[00119]

[00119] Targetedinsertion Targeted insertion of of the the rearranged humangermline rearranged human germline Vx1-39JK5 Vk1-39Jk5 region region intointo

BACDNA BAC DNAwaswas confirmed confirmed by polymerase by polymerase chain chain reaction reaction (PCR) (PCR) using primers using primers locatedlocated at at sequenceswithin sequences withinthe therearranged rearrangedhuman human germline germline light light chain chain region. region. Briefly,the Briefly, theintron intron sequence3'3'to sequence the mouse to the mouseV3-7 Vx3-7 leader leader sequence sequence was confirmed was confirmed with primers ULC-m1FULC-m1F with primers (AGGTGAGGGT ACAGATAAGT (AGGTGAGGGT ACAGATAAGT GTTATGAG; GTTATGAG; SEQ IDSEQ ID NO:2) NO:2) and ULC-mlR and ULC-m1R

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(TGACAAATGC CCTAATTATA GTGATCA; SEQ IDThe open The NO:3). open frame reading of frame of the 2023202781 04 May 2023

(TGACAAATGC CCTAATTATA GTGATCA; SEQ ID NO:3). reading the rearranged human rearranged human germline germline VK1-39JK5 Vk1-39Jk5 region region was confirmed was confirmed with primers with primers 1633-h2F 1633-h2F

(GGGCAAGTCA (GGGCAAGTCA GAGCATTAGC GAGCATTAGC A; SEQ A; SEQ ID NO:4) ID NO:4) andand 1633-h2R 1633-h2R (TGCAAACTGG (TGCAAACTGG ATGCAGCATA ATGCAGCATA G; SEQ G; SEQ ID NO:5). ID NO:5). The The neomycin neomycin cassette cassette was was confirmed confirmed with with primersneoF primers neoF (GGTGGAGAGG (GGTGGAGAGG CTATTCGGC; CTATTCGGC; SEQNO:6) SEQ ID ID NO:6) and and neoR neoR (GAACACGGCG (GAACACGGCG GCATCAG; GCATCAG; SEQIDIDNO:7). SEQ Targeted NO:7).Targeted BAC BAC DNAthen DNA was wasused thentoused to electroporate electroporate mouse mouse ES cells ES to cells to created modified created modified ES EScells cells for for generating chimeric mice generating chimeric mice that that express expressaa rearranged rearrangedhuman human germline VK1-39JK5 germline Vk1-39Jk5 region. region.

[00120]

[00120] Positive ES Positive cell clones ES cell clones were confirmed by were confirmed byTAQMAN TAQMAN screening screening and TM and karyotyping using karyotyping using probes probesspecific specific for for the the engineered VK1-39JK5 engineered Vk1-39Jk5 lightchain light chainregion regioninserted inserted into the into theendogenous endogenouslocus. locus.Briefly, Briefly,probe neoPneoP probe (TGGGCACAAC (TGGGCACAAC AGACAATCGG AGACAATCGG CTG;CTG; SEQIDIDNO:8) SEQ NO:8) which which binds binds within within theneomycin the neomycin marker marker gene, gene, probeprobe ULC-m1P ULC-m1P

(CCATTATGAT (CCATTATGAT GCTCCATGCC GCTCCATGCC TCTCTGTTC; TCTCTGTTC; SEQ which SEQ ID NO:9) ID NO:9) which binds bindsthe within within the intron intron sequence 3' sequence 3'to tothe themouse mouseV3-7 leader sequence, 3-7 leader sequence, and and probe probe 1633h2P (ATCAGCAGAA 1633h2P (ATCAGCAGAA

ACCAGGGAAA SEQ IDSEQ GCCCCT; ACCAGGGAAA GCCCCT; ID NO:10) NO:10) which within which binds binds within the rearranged the rearranged human human germline Vk1-39Jk5 germline open Vx1-39J5open reading reading frame. frame. Positive Positive ES cell ES cell clones clones werewere thenthen used used to implant to implant

femalemice female mice to to give give riserise to atolitter a litterofofpups pups expressing expressing the germline the germline Vk1-39Jk5 light chain light V1-39JK5 chain region. region.

[00121]

[00121] Alternatively, ES Alternatively, ES cells cellsbearing bearingthe therearranged rearranged human germlineVk1-39Jk5 human germline V1-39JK5 light light chain regionare chain region aretransfected transfected withwith a constuct a constuct that expresses that expresses FLP toin remove FLP in order order the to remove the FRTedneomycin FRTed neomycin cassette cassette introduced introduced by the by the targeting targeting construct. construct. Optionally, Optionally, thethe neomycin neomycin

cassette is cassette is removed bybreeding removed by breedingtotomice micethat thatexpress expressFLP FLP recombinase recombinase (e.g., (e.g., US US 6,774,279). Optionally, 6,774,279). Optionally, the the neomycin neomycincassette cassetteisisretained retained inin the the mice mice

C. Rearranged Human C. Rearranged Human Germline Germline VK3-20JK1 V3-20J1 Targeting Targeting VectorVector (Figure (Figure 2) 2)

[00122]

[00122] In aa similar In similar fashion, fashion,ananengineered engineered lightlight chain chain locuslocus expressing expressing a rearranged a rearranged

humangermline human germline region region VK3-20J1 3-20J1 was was made made using using a targeting a targeting constructconstruct including, including, from 5' from 5' to 3', to 3',a a5'5' homology homology arm containing sequence arm containing sequence5'5'to the endogenous to the endogenous mouseK mouse light K light chain chain

locus obtained locus from mouse obtained from mouseBACBAC clone clone 302g12, 302g12, a FRTed a FRTed neomycin neomycin resistance resistance gene, a gene, a genomicsequence genomic sequence including including thethe human human 3-15VK3-15 promoter, promoter, leader sequence a leadera sequence of the of the mouse mouse VK3-7 3-7 variable variable gene gene segment, segment, an intron an intron sequence sequence of theofmouse the mouse V3-7 variable 3-7 variable gene gene segment, segment, ananopen openreading reading frame frame of of a rearranged a rearranged human human germline germline V3-20Jk1 region, region, V3-20JK1 a a genomicsequence genomic sequence containing containing a portion a portion of of thethehuman humanJK-CKintron, JK-CK intron, and and 3' homology a 3'a homology arm arm containing sequence3'3'ofofthe containing sequence the endogenous endogenous mouse mouse JK5 gene JK5 gene segment segment obtained obtained from mouse from mouse

BACclone BAC clone 254m04 254m04(Figure (Figure2, 2, middle). middle). The The sequence sequence of ofthe theengineered human engineered humanV3-20JK1 V3-20Jk1

locus is locus is shown in SEQ shown in SEQIDIDNO:11. NO:11.

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[00123] Targeted insertion insertion of of the the rearranged humangermline germline V3-20JK1 region into 2023202781 04 May 2023

[00123] Targeted rearranged human V3-20Jk1 region into

BAC DNA BAC DNA waswas confirmed confirmed by polymerase by polymerase chain chain reaction reaction (PCR) (PCR) using primers using primers locatedlocated at at sequenceswithin sequences withinthe therearranged rearrangedhuman human germline germline light light VK3-20JK1 Vk3-20Jk1 chainchain region. region. Briefly, Briefly, the the

intron intron sequence 3'to sequence 3' to the the mouse V3-7 mouse 3-7 leader leader sequence sequence was confirmed was confirmed with primers with primers ULC- ULC m1F (SEQ m1F (SEQIDID NO:2) NO:2) and and ULC-m1R ULC-m1R (SEQ (SEQ ID ID NO:3).TheThe NO:3). open open reading reading frameofofthe frame the rearranged human rearranged human germline germline VK3-20J1 Vk3-20Jk1 region region was confirmed was confirmed with primers with primers 1635-h2F 1635-h2F

(TCCAGGCACC CTGTCTTTG; (TCCAGGCACC CTGTCTTTG; SEQ SEQ ID NO:12) ID NO:12) andand 1635-h2R(AAGTAGCTGC 1635-h2R (AAGTAGCTGC TGCTAACACT TGCTAACACT CTGACT; CTGACT; SEQ SEQ ID ID NO:13). NO:13). The neomycin The neomycin cassette cassette was confirmed was confirmed with with primers primers neoF neoF (SEQ ID NO:6) (SEQ ID NO:6) and and neoR neoR (SEQ ID NO:7). (SEQ ID NO:7). Targeted Targeted BAC DNAwas BAC DNA wasthen thenused used to electroporate to electroporate mouse EScells mouse ES cellsto to created created modified modifiedESEScells cellsfor for generating generating chimeric chimeric mice mice that express that the rearranged express the rearranged human human germline germline V3-20JK1 3-20J1 light chain. light chain.

[00124]

[00124] Positive Positive ES cell clones ES cell clones were confirmed by were confirmed byTaqman Taqman TM screening screening and and karyotyping using probes karyotyping using probesspecific specific for for the the engineered light light V3-20Ji1 engineered 3-20J1 chain chain region region inserted inserted

into into the the endogenous light chain endogenous K - light locus. Briefly, chain locus. Briefly, probe probe neoP (SEQIDIDNO:8) neoP (SEQ NO:8) which which binds binds

within the within the neomycin markergene, neomycin marker gene,probe probe ULC-mlP ULC-m1P (SEQ (SEQ ID which ID NO:9) NO:9) binds whichwithin binds the within the mouse Vi3-7 mouse leader sequence, V3-7 leader sequence,andand probe 1635h2P probe (AAAGAGCCAC 1635h2P (AAAGAGCCACCCTCTCCTGC CCTCTCCTGC AGGG;SEQ AGGG; SEQID ID NO:14)which NO:14) whichbinds bindswithin within the the human 3-20J1 openopen human VK3-20Ji1 reading reading frame. frame.

Positive ES Positive cell clones ES cell clones were were then used to then used to implant implant female female mice. mice. A Alitter litter of of pups pups expressing expressing

the human the germlineVk3-20Jk1 human germline VK3-20JK1 light light chain chain region. region.

[00125]

[00125] Alternatively, ES Alternatively, ES cells cellsbearing bearinghuman germine3-20J1 human germline light light VK3-2OJK1 chainchain region region

can be transfected can be transfected with with aa constuct constuct that that expresses FLPininoder expresses FLP odertotoremove removethe theFRTed FRTed neomycincassette neomycin cassetteintroduced introducedbybythe thetargeting targetingconsruct. consruct.Optionally, Optionally,the theneomycin neomycin cassette cassette

maybeberemoved may removedby by breeding breeding to to mice mice that that express express FLP FLP recombinase recombinase (e.g.,(e.g., US 6,774,279). US 6,774,279).

Optionally, theneomycin Optionally, the neomycin cassette cassette is retained is retained in the in the mice. mice.

D. Rearranged D. Rearranged Human Human Germline Germline VpreBJ%5 VpreBJX5 Targeting Targeting Vector Vector (Figure (Figure 3) 3)

[00126]

[00126] In aa similar In similar fashion, anengineered fashion, an engineered lightlight chain chain locuslocus expressing expressing a rearranged a rearranged

human germline human germline VpreBJ5 VpreBJX5 region region was was made made using using a targeting a targeting construct construct including, including, from from 5' to5' to

3', aa5'5'homology 3', arm containing homology arm containing sequence sequence5' 5'to theendogenous to the endogenous mouse mouse K light K light chain chain locus locus

obtained from mouse obtained from mouseBACBAC clone clone 302g12, 302g12, a FRTed a FRTed neomycin neomycin resistance resistance gene, angene, an genomic genomic

sequence including sequence including the thehuman humanVK3-15 promoter, aa leader 3-15 promoter, leader sequence sequence of ofthe themouse mouse V3-7 V3-7

variable gene variable segment,ananintron gene segment, intronsequence sequenceof of thethemouse mouse 3-7 VK3-7 variable variable gene segment, gene segment, an an open reading open readingframe frameofofa arearranged rearrangedhuman human germline germline VpreBJk5 VpreBJX5 region, region, a genomic a genomic sequence sequence

containing aa portion containing portion of of the thehuman intron, and Jx-CKintron, human JK-CK and aa 3' 3' homology homologyarm armcontaining containing sequence sequence

3'ofofthe 3' endogenous the endogenousmouse mouse JK5 gene segment J5 gene segment obtained obtained from from mouse BACclone mouse BAC clone 254m04 254m04

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(Figure 3, 3, middle). middle). The sequenceofofthe theengineered engineeredhuman human VpreBJk5 locus locus is shown in 2023202781 04 2023

(Figure The sequence VpreBJX5 is shown in

SEQ ID SEQ ID NO:15. NO:15.

[00127]

[00127] Targeted insertion Targeted insertion of of the the rearranged humangermline rearranged human germline VpreBJk5 VpreBJX5 region region into into May BACDNA BAC DNAwaswas confirmed confirmed by polymerase by polymerase chain chain reaction reaction (PCR) (PCR) using primers using primers locatedlocated at at sequenceswithin sequences withinthe therearranged rearrangedhuman human germline germline VpreBJk5 VpreBJX5 regionregion light light chainchain region. region.

Briefly, the Briefly, theintron sequence intron sequence 3'3'totothe mouse the mouse VK3-7 leader 3-7 leader sequence sequence was was confirmed confirmed with with primers ULC-m1F primers (SEQIDIDNO:2) ULC-m1F (SEQ NO:2)and and ULC-m1R ULC-mIR (SEQ (SEQ ID ID NO:3).TheThe NO:3). open open reading reading frame frame of the of the rearranged humangermline rearranged human germline VpreBJk5 VpreBJX5 region region was was confirmed confirmed with primers with primers 1616-h1F 1616-h1F

(TGTCCTCGGCCCTTGGA; (TGTCCTCGGC CCTTGGA; SEQSEQ ID NO:16) ID NO:16) andand1616-h1R 1616-h1R(CCGATGTCAT (CCGATGTCAT GGTCGTTCCT; GGTCGTTCCT; SEQ SEQ ID NO:17). ID NO:17). The The neomycin neomycin cassette cassette was was confirmed confirmed with with primersneoF primers neoF (SEQ ID (SEQ ID NO:6) NO:6) and and neoR neoR (SEQ (SEQID ID NO:7). NO:7). Targeted Targeted BAC BACDNA DNA was was thenused then usedtoto electroporate mouseESEScells electroporate mouse cellstotocreated createdmodified modifiedESES cellsfor cells forgenerating generatingchimeric chimericmice micethat that expressthe express the rearranged rearrangedhuman human germline germline VpreBJk5 VpreBJX5 lightlight chain. chain.

[00128]

[00128] Positive Positive ES cell clones ES cell clones are are confirmed by TAQMAN confirmed by TAQMAN TM screeningand screening and

karyotyping using karyotyping using probes probesspecific specific for for the the engineered VpreBJk5 engineered VpreBJX5 lightchain light chainregion regioninserted inserted into the into theendogenous light chain endogenous KKlight chain locus. Briefly, probe locus. Briefly, probe neoP (SEQIDIDNO:8) neoP (SEQ NO:8) which which binds binds

within the within the neomycin markergene, neomycin marker gene,probe probe ULC-m1P ULC-m1P (SEQ (SEQ ID ID which NO:9) NO:9)binds whichwithin binds the within the mouse IgVk3-7 mouse IgVK3-7 leader leader sequence, sequence, and and probe probe 1616h1P 1616h1P (ACAATCCGCC TCACCTGCAC (ACAATCCGCC TCACCTGCAC

CCT; SEQ CCT;SEQ ID ID NO:18) NO:18) which which binds binds within within the the human human VpreBJ5 VpreBJX5 open reading open reading frame. Positive frame. Positive

EScell ES cell clones clonesareare then then usedused to implant to implant femalefemale mice to mice to give give rise to a rise to aof litter litter pups of pups expressing expressing

germlinelight a germline a lightchain chain region. region.

[00129]

[00129] Alternatively, ES Alternatively, ES cells cellsbearing bearingthe therearranged rearranged human germlineVpreBJX5 human germline VpreBJ5 light light

chain region chain region are are transfected transfected with with aa construct construct that thatexpresses expresses FLP in order FLP in order to to remove the remove the

FRTedneomycin FRTed neomycin cassette cassette introduced introduced by the by the targeting targeting consruct. consruct. Optionally, Optionally, thethe neomycin neomycin

cassette is removed cassette is bybreeding removed by breedingtotomice micethat thatexpress expressFLP FLP recombinase recombinase (e.g., (e.g., US US

6,774,279). Optionally, 6,774,279). Optionally, the the neomycin neomycincassette cassetteisisretained retained inin the the mice. mice.

Example 3. Generation Example 3. GenerationofofMice Mice expressing expressingaa single single rearranged humangermline rearranged human germlinelight light chain chain

[00130]

[00130] Targeted ES Targeted EScells cellsdescribed describedabove above were were used used as donor as donor ES cells ES cells and and introduced into introduced into an an 8-cell 8-cellstage stage mouse embryoby by mouse embryo theVELOCIMOUSE® the VELOCIMOUSE@ method method (see, (see, e.g., e.g., US Pat. No. US Pat. No. 7,294,754 7,294,754and andPoueymirou Poueymirou et al. et al. (2007) (2007) F0 FO generation generation micemice thatthat are are essentially essentially

fully derived fully derivedfrom fromthe thedonor donor gene-targeted EScells gene-targeted ES cells allowing allowing immediate immediatephenotypic phenotypic analyses analyses

Nature Biotech. 25(1):91-99. Nature Biotech. 25(1):91-99. VELOCIMICE® VELOCIMICE@ independently independently bearingbearing an engineered an engineered human human germline Vk1-39Jk5 germline VK1-39JK5 lightchain light chainregion, region, aa 3-20J1 light light Vx3-20J1 chainchain region region or a or a VpreBJk5 VpreBJX5 light light chainregion chain regionareare identified identified by by genotyping genotyping using using a modification a modification of alleleofassay allele(Valenzuela assay (Valenzuela et et

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aL, supra) supra)that thatdetects detectsthe presence presence of ofthe theunique uniquerearranged rearranged human germlinelight light chain chain 2023202781 04 2023

al., the human germline

region. region.

[00131]

[00131] Pupsare Pups are genotyped genotypedandand a pup a pup heterozygous heterozygous for the for the unique unique rearranged rearranged May humangermline human germlinelight lightchain chainregion regionare are selected selectedfor for characterizing characterizing expression expressionofof the the rearranged human rearranged human germline germline lightchain light chainregion. region.

Example 4. Example 4. Breeding Breedingof of Mice Mice expressing expressingaa single single rearranged humangermline rearranged human germlinelight light chain chain A. A. Endogenous Knockout IgXKnockout Endogenous IgA (KO). (KO).

[00132]

[00132] To optimize the To optimize the usage usageofofthe the engineered engineeredlight light chain chain locus, locus, mice mice bearing bearingone one of the of the rearranged humangermline rearranged human germline lightchain light chainregions regionsare arebred bredtotoanother anothermouse mouse containing containing

a deletionininthe a deletion theendogenous endogenous k light 2 light chainchain locus.locus. In this In this manner, manner, theobtained the progeny progeny obtained will will express,asastheir express, theironly only lightchain, light chain, thethe rearranged rearranged human human germline germline light light chain chain region as region as described in described in Example Example2.2.Breeding Breeding is is performed performed by by standard standard techniques techniques recognized recognized in in the the art and, art and, alternatively, alternatively, bybya commercial a commercial breeder breeder (e.g., (e.g.,The TheJackson Laboratory). Mouse Jackson Laboratory). Mouse strains bearing strains bearing an an engineered light chain engineered light chain locus locus and deletion of and aa deletion of the theendogenous light endogenous 2 klight

chain locus chain locus are are screened screenedfor for presence presenceofofthe theunique uniquelight light chain region and chain region and absence absenceofof endogenous endogenous mouse mouse k light 2 light chains. chains.

B. Humanized B. Endogenous Humanized Endogenous Heavy Heavy Chain Chain Locus. Locus.

[00133]

[00133] Mice bearing Mice bearing an an engineered engineeredhuman human germline germline light light chain chain locus locus areare bred bred with with

mice that mice that contain a replacement contain a ofthe replacement of the endogenous endogenous mouse mouse heavyheavy chainchain variable variable gene gene locus locus

with with the the human heavychain human heavy chain variablegene variable gene locus locus (see (see US US 6,596,541; 6,596,541; the the VELOCIMMUNE@ VELOCIMMUNE®

mouse, Regeneron mouse, RegeneronPharmaceuticals, Pharmaceuticals, Inc.). Inc.). The TheVELOCIMMUNE@ mouse VELOCIMMUNE® mouse comprises comprises a a genomecomprising genome comprising human human heavyheavy chain chain variable variable regions regions operably operably linkedlinked to endogenous to endogenous

mouseconstant mouse constantregion regionloci locisuch suchthat that the the mouse mouseproduces produces antibodies antibodies comprising comprising a human a human

heavychain heavy chainvariable variable region region and andaamouse mouse heavy heavy chain chain constant constant region region in response in response to to antigenic antigenic stimulation. stimulation. The DNAencoding The DNA encoding thethe variable variable regions regions of of theheavy the heavy chains chains of of thethe

antibodies is antibodies is isolated isolatedand and operably operably linked linked to toDNA encodingthe DNA encoding thehuman human heavy heavy chain chain constant constant

regions. The regions. TheDNA DNAis is thenexpressed then expressed in cell in a a cellcapable capableofofexpressing expressing thethe fully human fully human heavy heavy

chain ofthe chain of theantibody. antibody.

[00134]

[00134] Mice bearing aa replacement Mice bearing replacementofofthe theendogenous endogenous mouse mouse VH locus VH locus with the with the

humanVHVH human locus locus andand a single a single rearranged rearranged human human germline germline VL region VL region at theatendogenous the endogenous K K

light lightchain chainlocus locusare areobtained. obtained. Reverse chimericantibodies Reverse chimeric antibodiescontaining containingsomatically somaticallymutated mutated heavy chains(human heavy chains (humanVH VH and and mouse mouse CH) awith CH) with a single single human human light chain light chain (human(human VL and VL and

mouseCL) mouse CL)are areobtained obtained upon upon immunization immunization withwith an antigen an antigen of interest. of interest. VH and VH and VL VL

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nucleotide sequences sequences ofofB Bcells cells expressing expressingthe theantibodies antibodiesare areidentified identified and fully human 2023202781 04 May 2023

nucleotide and fully human

antibodies are antibodies madebybyfusion are made fusionthe theVHVHand andVLVL nucleotide nucleotide sequences sequences to human to human CL and CH andCH CL nucleotide sequences nucleotide sequences inina asuitable suitable expression expressionsystem. system.

Example 5. Generation Example5. Generation ofofAntibodies Antibodies from from Mice Mice Expressing ExpressingHuman Human Heavy Heavy Chains Chains and and aa Rearranged HumanGermline Rearranged Human Germline Light Light Chain Chain Region Region

[00135]

[00135] After After breeding mice that breeding mice that contain the engineered contain the human engineered human lightchain light chainregion regiontoto various desired various desired strains strains containing containing modifications modifications and deletions of and deletions of other other endogenous endogenous IgIgloci loci (as described (as in Example described in 4), selected Example 4), selected mice micecan canbebeimmunized immunized withwith an antigen an antigen of interest. of interest.

[00136]

[00136] Generally, a aVELOCIMMUNE@ Generally, mouse VELOCIMMUNE® mouse containing containing oneone of of thesingle the single rearranged human rearranged human germline germline lightchain light chainregions regionsisischallenged challengedwith withananantigen, antigen,and andlymphatic lymphatic cells (such cells (such as as B-cells) B-cells)are arerecovered recovered from from serum of the serum of the animals. Thelymphatic animals. The lymphaticcells cellsare are fused with fused with aa myeloma cellline myeloma cell line to to prepare immortal hybridoma prepare immortal hybridomacell celllines, lines, and such hybridoma and such hybridoma cell cell lines lines are screened are screened andand selected selected to identify to identify hybridoma hybridoma cellthat cell lines lines that antibodies produce produce antibodies containing humanheavy containing human heavy chain chain variables variables andand a rearranged a rearranged human human germline germline light light chains chains

which are which are specific specific to to the the antigen antigen used used for for immunization. DNAencoding immunization. DNA encoding thethe variable variable regions regions

of of the heavychains the heavy chains and and the light the light chainchain are isolated are isolated and tolinked and linked to desirable desirable isotypic constant isotypic constant

regions of regions of the the heavy chain and heavy chain and light light chain. chain. Due to the Due to the presence presenceofofthe the endogenous endogenous mouse mouse

sequences andanyany sequences and additionalcis-acting additional cis-actingelements elementspresent present in in theendogenous the endogenous locus, locus, the the

single single light lightchain chainofof each eachantibody antibodymay may be somatically mutated. be somatically Thisadds mutated. This addsadditional additional diversity to the diversity to the antigen-specific antigen-specific repertoire repertoire comprising comprising a single a single light and light chain chain and heavy diverse diverse heavy chain sequences.TheThe chain sequences. resultingcloned resulting cloned antibody antibody sequences sequences are subsequently are subsequently expressed expressed in in cell, such aa cell, suchas as aaCHO cell. Alternatively, CHO cell. Alternatively,DNA encodingthe DNA encoding theantigen-specific antigen-specific chimeric chimeric antibodiesororthethevariable antibodies variable domains domains of theoflight the light and chains and heavy heavyare chains are identified identified directly directly from from antigen-specific antigen-specific lymphocytes. lymphocytes.

[00137]

[00137] Initially, high Initially, high affinity affinitychimeric chimeric antibodies areisolated antibodies are isolated having having a human a human variable variable

region region and mouseconstant and aa mouse constant region.As As region. described described above, above, the the antibodies antibodies are are characterized characterized

andselected and selectedforfor desirable desirable characteristics, characteristics, including including affinity, affinity, selectivity, selectivity, epitope, epitope, etc. etc. The The mouseconstant mouse constantregions regionsare arereplaced replaced witha adesired with desiredhuman human constant constant region region to generate to generate the the fully human fully antibody containing human antibody containing aa somatically somatically mutated mutatedhuman human heavy heavy chain chain and and a single a single light light

chain derived chain derived from from aa rearranged rearrangedhuman human germline germline light light chain chain region region of of theinvention. the invention. Suitable human Suitable constantregions human constant regionsinclude, include,for for example examplewild-type wild-typeorormodified modifiedIgG1 IgG1or or IgG4. IgG4.

[00138]

[00138] Separatecohorts Separate cohortsofof VELOCIMMUNE® VELOCIMMUNE@ mice containing mice containing a replacement a replacement of the of the endogenous mouse endogenous mouse heavy heavy chainchain locuslocus with with humanhuman V, D,J and V, D, and gene segments gene Jsegments and a and a replacementofofthe replacement the endogenous endogenous mouse mouse K light K light chain chain locus locus with with either either thethe engineered engineered

germline germline Vk1-39Jk5 human VK1-39J5human light light chain chain region region or or thethe engineered engineered germline germline Vk3-20Jk1 human human V3-20JK1

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light chain chainregion region(described (described above) above) were immunized witha ahuman human cell-surface receptor 2023202781 04 May 2023

light were immunized with cell-surface receptor

protein (Antigen protein (AntigenE).E).Antigen Antigen E is E is administered administered directlydirectly onto theonto hindthe hindoffootpad footpad of six mice with mice with six consecutiveinjections consecutive injections every 3-4 days. every 3-4 days. Two Twototothree threemicrograms microgramsof of Antigen Antigen E are E are mixed withwith mixed 10 µg 10 pg of of CpG oligonucleotide(Cat CpG oligonucleotide (Cat##tlrl-modn tirl-modn -- ODN1826 oligonucleotide; ODN1826 oligonucleotide InVivogen, ; InVivogen, SanSan

Diego, CA) Diego, CA) and and2525µgpgofofAdju-Phos Adju-Phos (Aluminum (Aluminum phosphate phosphate gel adjuvant, gel adjuvant, Cat# Cat# H-71639-250; H-71639-250;

BrenntagBiosector, Brenntag Biosector, Frederikssund, Frederikssund, Denmark) Denmark) prior to injection. prior to injection. A total of A total six of six injections injections are are given priortotothe given prior thefinal final antigen antigenrecall, recall,which which is given is given 3-5 3-5 days days prior prior to sacrifice. to sacrifice. Bleeds Bleeds after after the 4th the 4th and 6th injection and 6th injection are arecollected collectedand andthe theantibody antibody immune responseisismonitored immune response monitoredby by a a standard antigen-specific immunoassay. standard antigen-specific immunoassay.

[00139]

[00139] Whena adesired When desiredimmune immune response response is achieved is achieved splenocytes splenocytes are harvested are harvested and and fusedwith fused withmouse mouse myeloma myeloma cells tocells to preserve preserve their viability their viability and form and form cell hybridoma hybridoma lines. cell lines. The hybridoma The hybridoma cell cell lines lines are screened are screened and selected and selected to cell to identify identify linescell thatlines that produce produce

Antigen E-specific Antigen E-specific common common lightchain light chainantibodies. antibodies.Using Using thistechnique this techniqueseveral severalanti-Antigen anti-Antigen E-specific common E-specific lightchain common light chain antibodies antibodies(i.e., (i.e., antibodies antibodies possessing humanheavy possessing human heavy chain chain

variable domains, variable the same domains, the samehuman human light light chain chain variabledomain, variable domain, andand mouse mouse constant constant

domains) areobtained. domains) are obtained.

[00140]

[00140] Alternatively, anti-Antigen Alternatively, anti-AntigenE common E common light antibodies light chain chain antibodies are are isolated isolated directly from directly fromantigen-positive antigen-positive B cells B cells without without fusion fusion to myeloma to myeloma cells, ascells, as described described in U.S. in U.S. 2007/0280945A1, 2007/0280945A1, hereinherein specifically specifically incorporated incorporated by reference by reference in its Using in its entirety. entirety. this Using this method, severalfully method, several fully human anti-AntigenE Ecommon human anti-Antigen common light light chain chain antibodies antibodies (i.e., antibodies (i.e., antibodies possessing human possessing human heavy heavy chain chain variable variable domains, domains, either either an engineered an engineered humanhuman V1-39JK5 Vk1-39Jk5

light lightchain chainor oran anengineered engineered human V3-20JK1 human Vk3-20Jk1 light light chain chain region,and region, andhuman human constant constant

domains)were domains) wereobtained. obtained.

[00141]

[00141] The biological properties The biological properties of of the theexemplary anti-Antigen EE common exemplary anti-Antigen common lightchain light chain antibodies generated antibodies generatedinin accordance accordance withthe with themethods methods of of thisExample this Example are are described described in detail in detail

in the in sectionsset the sections setforth forthbelow. below.

Example6. Example 6. Heavy HeavyChain ChainGene Gene Segment Segment Usage Usage in Antigen-Specific in Antigen-Specific Common Common Light Light Chain Antibodies Chain Antibodies

[00142]

[00142] To analyze To analyzethe the structure structure of of the the human anti-AntigenEEcommon human anti-Antigen common light light chain chain

antibodies produced, antibodies produced,nucleic nucleicacids acidsencoding encodingheavy heavy chain chain antibody antibody variable variable regions regions were were

cloned and cloned andsequenced. sequenced. From From the the nucleic nucleic acidacid sequences sequences and predicted and predicted amino amino acid acid sequences sequences ofofthe theantibodies, antibodies,gene geneusage usage waswas identified forthe identifiedfor theheavy heavychain chainvariable variableregion region (HCVR)ofofselected (HCVR) selectedcommon common light light chain chain antibodies antibodies obtained obtained from from immunized immunized

VELOCIMMUNE@ VELOCIMMUNE® mice containing mice containing either either the the engineered engineered human V1-39JK5 human Vk1-39Jk5 light chainlight or chain or engineered human engineered human VK3-20J1 3-20J1 light region. light chain chain region. ResultsResults areinshown are shown Tablesin 3Tables and 4,3 and 4,

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which demonstrate which demonstratethat thatmice miceaccording according to to theinvention the inventiongenerate generate antigen-specificcommon antigen-specific common light lightchain chainantibodies antibodiesfrom from aavariety varietyofof human human heavy chain gene heavy chain genesegments, segments,duedue to to a variety a variety

of of rearrangements, when rearrangements, when employing employing either either a mouse a mouse thatthat expresses expresses a light a light chain chain fromfrom onlyonly a a May humanVk1-39- human or or VO1-39- a human a human V3-20-derived V3-20-derived light light chain. chain. HumanHuman V gene VH gene of segments segments the 2, of the 2, 3, 3, 4, 4, and and 55 families familiesrearranged rearranged with with aa variety varietyofof human human segmentsand DH segments DH and human human JH JH

segments to yield segments to yield antigen-specific antigen-specific antibodies. antibodies.

[00143]

[00143]

Table 33 Table

Vri-39Jw5 VK1-39JK5 Common Common Light Light Chain Chain Antibodies Antibodies HCVR HCVR Antibody Antibody VH VH DH DH JH

2952 2952 2-5 2-5 6-6 6-6 1 1

3022 3022 3-23 3-23 3-10 3-10 4 4 3028 3028 3-23 3-23 3-3 3-3 4 4 2955 2955 3-30 3-30 6-6 6-6 1 1

3043 3043 3-30 3-30 6-6 6-6 33 3014 3014 3-30 3-30 1-7 1-7 4 4 3015 3015 3-30 3-30 1-7 1-7 4 4 3023 3023 3-30 3-30 1-7 1-7 4 4 3024 3024 3-30 3-30 1-7 1-7 4 4 3032 3032 3-30 3-30 1-7 1-7 4 4 3013 3013 3-30 3-30 5-12 5-12 4 4 3042 3042 3-30 3-30 5-12 5-12 4 4 2985 2985 3-30 3-30 6-13 6-13 4 4 2997 2997 3-30 3-30 6-13 6-13 4 4 3011 3011 3-30 3-30 6-13 6-13 4 4 3047 3047 3-30 3-30 6-13 6-13 4 4 3018 3018 3-30 3-30 6-6 6-6 4 4 2948 2948 3-30 3-30 7-27 7-27 4 4 2987 2987 3-30 3-30 7-27 7-27 4 4 2996 2996 3-30 3-30 7-27 7-27 4 4 3005 3005 3-30 3-30 7-27 7-27 4 4 3012 3012 3-30 3-30 7-27 7-27 4 4 3020 3020 3-30 3-30 7-27 7-27 4 4 3021 3021 3-30 3-30 7-27 7-27 4 4 3025 3025 3-30 3-30 7-27 7-27 4 4 3030 3030 3-30 3-30 7-27 7-27 4 4

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3036 3-30 7-27 4 2023202781 04 2023

3036 3-30 7-27 4 2982 2982 3-30 3-30 3-22 3-22 5 5 2949 2949 3-30 3-30 6-6 6-6 55 May 2950 2950 3-30 3-30 6-6 6-6 55 2954 2954 3-30 3-30 6-6 6-6 55 2978 2978 3-30 3-30 6-6 6-6 55 3016 3016 3-30 3-30 6-6 6-6 55 3017 3017 3-30 3-30 6-6 6-6 55 3033 3033 3-30 3-30 6-6 6-6 55 3041 3041 3-30 3-30 6-6 6-6 55 3004 3004 3-30 3-30 7-27 7-27 5 5

3010 3010 4-59 4-59 3-16 3-16 33 3019 3019 4-59 4-59 3-16 3-16 3 3 2964 2964 4-59 4-59 3-22 3-22 3 3

3027 3027 4-59 4-59 3-16 3-16 44 3046 3046 5-51 5-51 5-5 5-5 3 3

[00144]

[00144]

Table Table 4 4

VK3-2OJxl V3-20J1 Common Light Common Light Chain Chain Antibodies Antibodies HCVR HCVR Antibody Antibody VH VH DH DH JH 2968 2968 4-39 4-39 1-26 1-26 3 3 2975 2975 5-51 5-51 6-13 6-13 5 5

2972 2972 5-51 5-51 3-16 3-16 6 6

Example 7. Example Determination of 7. Determination of Blocking Ability of BlockingAbility ofAntigen-Specific Antigen-SpecificCommon Light Common Light Chain TM Chain Antibodies Antibodiesbyby Luminex Luminex Assay Assay

[00145]

[00145] Ninety-eight Ninety-eight human common human common light light chain chain antibodies antibodies raised raised against against Antigen Antigen E E

weretested were tested forfor theirability their abilitytotoblock block binding binding of Antigen of Antigen E's natural E's natural ligandligand (Ligand(Ligand Y) to Y) to Antigen EE in Antigen in aa bead-based bead-basedassay. assay.

[00146]

[00146] The extracellular The extracellular domain (ECD)ofofAntigen domain (ECD) AntigenE Ewas was conjugated conjugated to two to two mycmyc

epitope tags and epitope tags and aa 6X histidine tag 6X histidine tag (Antigen (Antigen E-mmH) and E-mmH) and amine-coupled amine-coupled to carboxylated to carboxylated

microspheres atataa concentration microspheres concentrationofof20 20µg/mL pg/mLininMES MES buffer.TheThe buffer. mixture mixture was was incubated for for incubated two hours two hours at at room roomtemperature followedby by temperaturefollowed bead bead deactivation deactivation with with 1M 1M Tris pH pH Tris 8.0 8.0 followed followed

by by washing washing inin PBS with0.05% PBSwith 0.05% (v/v)Tween-20. (v/v) Tween-20. The The were were beadsbeads then blocked then blocked with PBS with PBS

(Irvine Scientific, (Irvine Scientific,Santa Ana, Santa Ana,CA) CA)containing containing2% (w/v) BSA 2% (w/v) (Sigma-AldrichCorp., BSA (Sigma-Aldrich Corp.,St. St. Louis, Louis,

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MO). InIn aa 96-well MO). 96-well filter filter plate, plate,supernatants supernatantscontaining containingAntigen Antigen E-specific E-specificcommon light chain common light chain

antibodies, antibodies, were diluted 1:15 were diluted 1:15 in in buffer. buffer.AAnegative negative control controlcontaining containingaamock mock supernatant supernatant

with the with the same mediacomponents same media components as for as for the the antibody antibody supernatant supernatant was was prepared. prepared. Antigen Antigen E- E May labeled beads labeled beadswere wereadded addedto to thesupernatants the supernatants andand incubated incubated overnight overnight at 4°C. at 4°C. Biotinylated Biotinylated-

Ligand YY protein Ligand protein was wasadded addedtotoa afinal final concentration concentration of of 0.06 0.06 nM nM and andincubated incubatedfor fortwo twohours hours at room at temperature. Detection room temperature. Detectionofofbiotinylated-Ligand biotinylated-LigandY Ybound boundto to Antigen Antigen E-myc-myc-6His E-myc-myc-6His

labeled beads labeled beads was wasdetermined determined with with R-Phycoerythrin R-Phycoerythrin conjugated conjugated to Streptavidin to Streptavidin (Moss (Moss Inc, Inc,

Pasadena,MD) Pasadena, MD) followed followed by by measurement measurement in a LuminexTM in a Luminex flow cytometry-based flow cytometry-based analyzer. analyzer.

BackgroundMean Background Mean Fluorescence Fluorescence Intensity Intensity (MFI)(MFI) of aofsample a sample without without Ligand Ligand Y wasY was subtracted from all subtracted from all samples. Percentblocking samples. Percent blockingwas wascalculated calculatedby by divisionofofthe division the background- background subtracted MFI of subtracted MFI of each eachsample samplebyby theadjusted the adjusted negative negative controlvalue, control value,multiplying multiplyingbyby100 100 andsubtracting and subtractingthethe resulting resulting value value from from 100. 100.

[00147]

[00147] In In aa similar similarexperiment, experiment, the the same 98 human same 98 humancommon common lightlight chain chain antibodies antibodies

raised against raised againstAntigen Antigen E were E were testedtested for ability for their their ability to block to block binding binding of Antigen of Antigen E to E to Ligand Ligand Y-labeled beads. Y-labeled beads.

[00148]

[00148] Briefly, Briefly,Ligand LigandYY was amine-coupledtotocarboxylated was amine-coupled carboxylatedmicrospheres microspheres at a at a

concentration of concentration of 20 diluted in pg/mLdiluted 20 µg/mL in MES MESbuffer. buffer. The Themixture mixtureand and incubated incubated twotwo hours hours at at roomtemperature room temperaturefollowed followedbybydeactivation deactivationofofbeads beads with1M1M with Tris Tris pH pH 8 then 8 then washing washing in PBS in PBS

with 0.05% with (v/v) Tween-20. 0.05% (v/v) The Tween-20. The beads beads werewere then then blocked blocked with with PBS (Irvine PBS (Irvine Scientific, Scientific, Santa Santa

Ana, CA) Ana, CA) containing containing2%2%(w/v) (w/v)BSA BSA (Sigma-Aldrich (Sigma-Aldrich Corp., Corp., St. St. Louis, Louis, MO). MO). In aIn96-well a 96-well filter filter

plate, supernatants plate, containing Antigen supernatants containing Antigen E-specific E-specific common lightchain common light chainantibodies antibodieswere were diluted 1:15 diluted 1:15 in inbuffer. buffer.A Anegative negative control controlcontaining containinga amock mock supernatant with the supernatant with the same same

media components media componentsas as forfor thethe antibody antibody supernatant supernatant waswas prepared. prepared. A biotinylated-Antigen A biotinylated-Antigen E- E mmHwaswas mmH added added to atofinal a final concentration concentration of of 0.42nMnM 0.42 andand incubated incubated overnight overnight at 40C. at 4°C. Ligand Ligand

Y-labeled beads Y-labeled beadswere werethen thenadded added to to thethe antibody/Antigen antibody/Antigen E mixture E mixture and and incubated incubated for for two two hours at hours at room temperature.Detection room temperature. of of Detection biotinylated-AntigenE-mmH biotinylated-Antigen E-mmH bound bound to Ligand to Ligand Y- Y beadswas beads wasdetermined determined with with R-Phycoerythrin R-Phycoerythrin conjugated conjugated to Streptavidin to Streptavidin (Moss (Moss Inc, Inc,

Pasadena,MD) Pasadena, MD) followed followed by by measurement measurement in a LuminexTM in a Luminex flow cytometry-based flow cytometry-based analyzer. analyzer.

BackgroundMean Background Mean Fluorescence Fluorescence Intensity Intensity (MFI)(MFI) of aofsample a sample without without Antigen Antigen E wasE was subtracted from all subtracted from all samples. Percentblocking samples. Percent blockingwas was calculatedby by calculated divisionofofthe division the background- background subtracted MFI ofof each subtracted MFI eachsample sampleby by theadjusted the adjusted negative negative controlvalue, control value,multiplying multiplyingbyby100 100 and subtracting and subtracting thethe resulting resulting value value from from 100. 100.

[00149]

[00149] Tables 55 and Tables and66show showthe thepercent percentblocking blockingfor forall all 98 98 anti-Antigen anti-Antigen EE common common light lightchain chainantibodies antibodiestested testedininboth bothLuminexTM assays. Luminex assays. ND: ND: not not determined determined underunder current current

experimental conditions. experimental conditions.

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[00150] In the the first LuminexTM experiment experimentdescribed described above, 80 common common lightchain chain 2023202781 04 May 2023

[00150] In first Luminex TM above, 80 light

antibodiescontaining antibodies containing thethe VK1-39J5 Vk1-39Jk5 engineered engineered light light chain werechain were tested tested for their for their ability to ability to block Ligand YY binding block Ligand binding to to Antigen Antigen E-labeled E-labeled beads. beads.OfOf these these 80 80 common common lightlight chain chain

antibodies, 68 antibodies, 68 demonstrated >50% demonstrated >50% blocking, blocking, while while 12 12 demonstrated demonstrated <50% <50% blocking blocking (6 at (6 at 25- 25 50%blocking 50% blockingand and6 6atat<25% <25% blocking).ForFor blocking). thethe 18 18 common common light light chain chain antibodies antibodies containing containing

the Vx3-20JK1 the engineered Vk3-20Jk1 engineered lightchain, light chain,1212demonstrated demonstrated >50% >50% blocking, blocking, whilewhile 6 6 demonstrated<50% demonstrated <50% blocking blocking (3 at (3 at 25-50% 25-50% blocking blocking and and at <25% 3 at 3<25% blocking) blocking) of Ligand of Ligand Y Y binding binding to to Antigen Antigen E-labeled beads. E-labeled beads.

[00151]

[00151] In the In thesecond secondLuminexTM described above, experiment described Luminex experiment above, the thesame same 80 80common common light chain light antibodiescontaining chain antibodies containing the the VK1-39J5 Vk1-39Jk5 engineered engineered light chainlight werechain testedwere tested for their for their ability totoblock ability binding block of of binding Antigen E to Antigen Ligand E to Y-labeled Ligand Y-labeledbeads. beads. Of Of these these 80 80 common light common light

chain antibodies, chain antibodies, 36 36 demonstrated >50% demonstrated >50% blocking, blocking, while while 44 44 demonstrated demonstrated <50% <50% blocking blocking

(27 (27 at at 25-50% blockingand 25-50% blocking and1717atat<25% <25% blocking).ForFor blocking). thethe 18 18 common common light light chain chain antibodies antibodies

containing the containing the VK3-20J1 engineered V3-20Jk1 engineered light light chain, chain, 1 demonstrated 1 demonstrated >50%>50% blocking, blocking, whilewhile 17 17 demonstrated<50% demonstrated <50% blocking blocking (5 at (5 at 25-50% 25-50% blocking blocking and and 12 at12<25% at <25% blocking) blocking) of Antigen of Antigen E E binding binding to to Ligand Ligand Y-labeled beads. Y-labeled beads.

[00152]

[00152] The data of The data of Tables Tables 55 and and66establish establish that that the the rearrangements described rearrangements described in in

Tables 33 and Tables and44generated generatedanti-Antigen anti-AntigenE-specific E-specificcommon common light light chain chain antibodies antibodies that that

blocked binding of blocked binding of Ligand Ligand YY to to its its cognate cognate receptor receptor Antigen with varying Antigen EE with varying degrees degreesofof efficacy, which efficacy, whichisisconsistent consistent with with the the anti-Antigen anti-Antigen E common E common light light chain chain antibodies antibodies of Tables of Tables 33 and comprisingantibodies and 44 comprising antibodieswith with overlapping overlappingand andnon-overlapping non-overlapping epitope epitope specificitywith specificity with respect respect totoAntigen AntigenE. E.

[00153]

[00153]

Table 5 Table 5

Vi1-39JK5 VK1-39JK5 Common Common Light Light Chain Chain Antibodies Antibodies

Antibody Antibody % Blocking of %Blocking of % of Blocking of % Blocking Antigen E-Labeled Beads Antigen E-Labeled Beads AntigenE EInInSolution Antigen Solution 2948 2948 81.1 81.1 47.8 47.8

2948G 2948G 38.6 38.6 ND ND 2949 2949 97.6 97.6 78.8 78.8

2949G 2949G 97.1 97.1 73.7 73.7

2950 2950 96.2 96.2 81.9 81.9

2950G 2950G 89.8 89.8 31.4 31.4

2952 2952 96.1 96.1 74.3 74.3

2952G 2952G 93.5 93.5 39.9 39.9

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04 May 2023

2954 2954 93.7 93.7 70.1 70.1

2954G 2954G 91.7 91.7 30.1 30.1 2955 2955 75.8 75.8 30.0 30.0 2955G 2955G 71.8 71.8 ND ND 2964 2964 92.1 92.1 31.4 31.4 2964G 2964G 94.6 94.6 43.0 43.0 2978 2978 98.0 98.0 95.1 95.1

2023202781 2978G 2978G 13.9 13.9 94.1 94.1 2982 2982 92.8 92.8 78.5 78.5 2982G 2982G 41.9 41.9 52.4 52.4 2985 2985 39.5 39.5 31.2 31.2 2985G 2985G 2.0 2.0 5.0 5.0 2987 2987 81.7 81.7 67.8 67.8 2987G 2987G 26.6 26.6 29.3 29.3 2996 2996 87.3 87.3 55.3 55.3 2996G 2996G 95.9 95.9 38.4 38.4 2997 2997 93.4 93.4 70.6 70.6 2997G 2997G 9.7 9.7 7.5 7.5 3004 3004 79.0 79.0 48.4 48.4 3004G 3004G 60.3 60.3 40.7 40.7 3005 3005 97.4 97.4 93.5 93.5 3005G 3005G 77.5 77.5 75.6 75.6 3010 3010 98.0 98.0 82.6 82.6 3010OG 3010G 97.9 97.9 81.0 81.0 3011 3011 87.4 87.4 42.8 42.8 3011 G 3011G 83.5 83.5 41.7 41.7 3012 3012 91.0 91.0 60.8 60.8 3012G 3012G 52.4 52.4 16.8 16.8 3013 3013 80.3 80.3 65.8 65.8 3013G 3013G 17.5 17.5 15.4 15.4 3014 3014 63.4 63.4 20.7 20.7 3014G 3014G 74.4 74.4 28.5 28.5 3015 3015 89.1 89.1 55.7 55.7 3015G 3015G 58.8 58.8 17.3 17.3 3016 3016 97.1 97.1 81.6 81.6 3016G 3016G 93.1 93.1 66.4 66.4 3017 3017 94.8 94.8 70.2 70.2 3017G 3017G 87.9 87.9 40.8 40.8 3018 3018 85.4 85.4 54.0 54.0 3018G 3018G 26.1 26.1 12.7 12.7

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3019 3019 99.3 99.3 92.4 92.4 3019G 3019G 99.3 99.3 88.1 88.1 3020 3020 96.7 96.7 90.3 90.3 May 3020G 3020G 85.2 85.2 41.5 41.5 3021 3021 74.5 74.5 26.1 26.1 3021 3021GG 81.1 81.1 27.4 27.4 3022 3022 65.2 65.2 17.6 17.6 3022G 3022G 67.2 67.2 9.1 9.1

3023 3023 71.4 71.4 28.5 28.5 3023G 3023G 73.8 73.8 29.7 29.7 3024 3024 73.9 73.9 32.6 32.6 3024G 3024G 89.0 89.0 10.0 10.0 3025 3025 70.7 70.7 15.6 15.6 3025G 3025G 76.7 76.7 24.3 24.3 3027 3027 96.2 96.2 61.6 61.6 3027G 3027G 98.6 98.6 75.3 75.3 3028 3028 92.4 92.4 29.0 29.0 3028G 3028G 87.3 87.3 28.8 28.8 3030 3030 6.0 6.0 10.6 10.6 3030G 3030G 41.3 41.3 14.2 14.2 3032 3032 76.5 76.5 31.4 31.4 3032G 3032G 17.7 17.7 11.0 11.0 3033 3033 98.2 98.2 86.1 86.1 3033G 3033G 93.6 93.6 64.0 64.0 3036 3036 74.7 74.7 32.7 32.7 3036G 3036G 90.1 90.1 51.2 51.2 3041 3041 95.3 95.3 75.9 75.9 3041 G 3041G 92.4 92.4 51.6 51.6 3042 3042 88.1 88.1 73.3 73.3 3042G 3042G 60.9 60.9 25.2 25.2 3043 3043 90.8 90.8 65.8 65.8 3043G 3043G 92.8 92.8 60.3 60.3

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[00154]

[00154]

Table 66 Table

V3-20Jic1 V3-20J1 Common Common Light Light Chain Chain Antibodies Antibodies

Antibody Antibody % Blocking of %Blocking of % Blocking % of Blockingof Antigen E-Labeled Antigen E-Labeled Beads Beads AntigenE EInInSolution Antigen Solution 2968 2968 97.1 97.1 73.3 73.3

2968G 2968G 67.1 67.1 14.6 14.6

2969 2969 51.7 51.7 20.3 20.3

2969G 2969G 37.2 37.2 16.5 16.5

2970 2970 92.2 92.2 34.2 34.2

2970G 2970G 92.7 92.7 27.2 27.2

2971 2971 23.4 23.4 11.6 11.6

2971G 2971G 18.8 18.8 18.9 18.9

2972 2972 67.1 67.1 38.8 38.8

2972G 2972G 64.5 64.5 39.2 39.2

2973 2973 77.7 77.7 27.0 27.0

2973G 2973G 51.1 51.1 20.7 20.7

2974 2974 57.8 57.8 12.4 12.4

2974G 2974G 69.9 69.9 17.6 17.6

2975 2975 49.4 49.4 18.2 18.2

2975G 2975G 32.0 32.0 19.5 19.5

2976 2976 1.0 1.0 1.0 1.0

2976G 2976G 50.4 50.4 20.4 20.4

Example 8. Example 8. Determination Determination of of Blocking Blocking Ability Ability of ofAntigen-Specific Antigen-SpecificCommon Light Common Light Chain Antibodies Chain Antibodies by by ELISA ELISA

[00155]

[00155] Human common Human common lightlight chain chain antibodies antibodies raised raised against against Antigen Antigen E were E were tested tested

for their for their ability abilityto toblock block Antigen Antigen E E binding binding to to a Ligand a Ligand Y-coated Y-coated surfacesurface in an in an ELISA ELISA assay. assay.

[00156]

[00156] Ligand YY was Ligand wascoated coatedonto onto96-well 96-wellplates platesatata aconcentration concentrationofof22µg/mL diluted pg/mLdiluted in PBS in andincubated PBS and incubatedovernight overnightfollowed followedbybywashing washing four four times times in in PBS PBS with with 0.05% 0.05% Tween Tween-

20. Theplate 20. The plate was wasthen thenblocked blockedwith PBS withPBS (IrvineScientific, (Irvine Scientific, Santa Santa Ana, Ana,CA) CA)containing containing0.5% 0.5% (w/v) BSA (w/v) (Sigma-AldrichCorp., BSA (Sigma-Aldrich Corp.,St. St. Louis, Louis, MO) MO)for forone onehour houratatroom roomtemperature. temperature. In In a a separate plate, supernatants separate plate, containing anti-Antigen supernatants containing anti-Antigen EE common common light light chainantibodies chain antibodies were were

diluted 1:10 diluted 1:10 in inbuffer. buffer.A Amock mock supernatant with the supernatant with the same samecomponents components of the of the antibodies antibodies waswas

used as aa negative used as negative control. control. Antigen AntigenE-mmH E-mmH (described (described above) above) was added was added to a final to a final

concentration of 0.150 concentration of nMand 0.150 nM andincubated incubatedforforone onehour houratatroom room temperature. temperature. The The

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antibody/Antigen E-mmH mixture was was thenthen added to plate the plate containing Ligand Y andY and 2023202781 04 2023

antibody/Antigen E-mmH mixture added to the containing Ligand

incubated for incubated for one hour at one hour at room roomtemperature. temperature.Detection Detection of of Antigen Antigen E-mmH E-mmH boundbound to Ligand to Ligand

wasdetermined Y was Y determinedwith withHorse-Radish Horse-Radish Peroxidase Peroxidase (HRP)(HRP) conjugated conjugated to anti-Penta-His to anti-Penta-His May antibody (Qiagen, Valencia, antibody (Qiagen, Valencia, CA) CA)and anddeveloped developed by by standard standard colorimetric colorimetric response response using using

tetramethylbenzidine (TMB) tetramethylbenzidine (TMB)substrate substrate(BD (BD Biosciences, Biosciences, SanSan Jose, Jose, CA) CA) neutralized neutralized by by sulfuric sulfuricacid. acid.Absorbance wasread Absorbance was readatatOD450 OD450forfor 0.10.1 sec.Background sec. Background absorbance absorbance of a of a

sample without Antigen sample without AntigenE Ewas was subtracted subtracted from from allallsamples. samples. Percent Percent blocking blocking was was calculated calculated

by division by division ofofthe thebackground-subtracted MFlofofeach background-subtracted MFI eachsample sampleby by thethe adjusted adjusted negative negative

control value,multiplying control value, multiplyingby by 100100 and and subtracting subtracting the resulting the resulting value value from 100.from 100.

[00157]

[00157] Tables 77 and Tables and88show showthe thepercent percentblocking blockingfor forall all 98 98 anti-Antigen anti-Antigen EE common common light chain light chainantibodies antibodiestested testedinin the ELISA the ELISA assay. assay. ND: not determined ND: not determinedunder undercurrent current experimental conditions. experimental conditions.

[00158]

[00158] As described As describedinin this this Example, of the Example, of the 80 80 common common lightchain light chainantibodies antibodies containingthe containing theVK1-39JK5 V1-39JK5 engineered engineered lighttested light chain chainfor tested their for theirtoability ability block to blockEAntigen Antigen E binding to binding to aa Ligand Ligand Y-coated surface, 22 Y-coated surface, 22 demonstrated demonstrated>50% >50% blocking, blocking, while while 58 58 demonstrated<50% demonstrated <50% blocking blocking (20 (20 at 25-50% at 25-50% blocking blocking andat38<25% and 38 at <25% blocking). blocking). For For the 18the 18 common common lightchain light chainantibodies antibodiescontaining containingthe theV3-20Jk1 VK3-20JK1 engineered engineered lightlight chain, chain, 1 1 demonstrated>50% demonstrated >50% blocking, blocking, while while 17 17 demonstrated demonstrated <50% <50% blocking blocking (5 at 25-50% (5 at 25-50% blocking blocking

and 12 and 12 at at <25% <25%blocking) blocking)ofofAntigen AntigenE Ebinding bindingtotoaaLigand LigandY-coated Y-coated surface. surface.

[00159]

[00159] Theseresults These results are are also also consistent consistent with with the the Antigen E-specific common Antigen E-specific light common light

chain antibody chain antibody pool pool comprising comprisingantibodies antibodieswith with overlapping overlappingand andnon-overlapping non-overlapping epitope epitope

specificity with specificity respecttotoAntigen with respect Antigen E. E.

[00160]

[00160]

Table Table 77

VKI-39JK5 VK1-39JK5 Common Light Chain Common Light Chain Antibodies Antibodies

Antibody %Blocking % of Blockingof Antibody Y Antigen Solution AntigenE EInInSolution 2948 2948 21.8 21.8

2948G 2948G 22.9 22.9

2949 2949 79.5 79.5

2949G 2949G 71.5 71.5

2950 2950 80.4 80.4

2950G 2950G 30.9 30.9

2952 2952 66.9 66.9

2952G 2952G 47.3 47.3

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2954 2954 55.9 55.9

2954G 2954G 44.7 44.7

2955 2955 12.1 12.1

2955G 2955G 25.6 25.6

2964 2964 34.8 34.8

2964G 2964G 47.7 47.7 2978 2978 90.0 90.0

2978G 2978G 90.2 90.2

2982 2982 59.0 59.0

2982G 2982G 20.4 20.4

2985 2985 10.5 10.5

2985G 2985G ND ND 2987 2987 31.4 31.4

2987G 2987G ND ND 2996 2996 29.3 29.3

2996G 2996G ND ND 2997 2997 48.7 48.7

2997G 2997G ND ND 3004 3004 16.7 16.7

3004G 3004G 3.5 3.5

3005 3005 87.2 87.2

3005G 3005G 54.3 54.3

3010 3010 74.5 74.5

3010G 3010G 84.6 84.6

3011 3011 19.4 19.4

3011G 3011G ND ND 3012 3012 45.0 45.0

3012G 3012G 12.6 12.6

3013 3013 39.0 39.0

3013G 3013G 9.6 9.6

3014 3014 5.2 5.2

3014G 3014G 17.1 17.1

3015 3015 23.7 23.7

3015G 3015G 10.2 10.2

3016 3016 78.1 78.1

3016G 3016G 37.4 37.4

3017 3017 61.6 61.6

3017G 3017G 25.2 25.2

3018 3018 40.6 40.6

3018G 3018G 14.5 14.5

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3019 3019 94.6 94.6

3019G 3019G 92.3 92.3

3020 3020 80.8 80.8

3020G 3020G ND ND 3021 3021 7.6 7.6

3021G 3021G 20.7 20.7

3022 3022 2.4 2.4

2023202781 3022G 3022G 15.0 15.0

3023 3023 9.1 9.1

3023G 3023G 19.2 19.2

3024 3024 7.5 7.5

3024G 3024G 15.2 15.2

3025 3025 ND ND 3025G 3025G 13.9 13.9

3027 3027 61.4 61.4

3027G 3027G 82.7 82.7

3028 3028 40.3 40.3

3028G 3028G 12.3 12.3

3030 3030 ND ND 3030G 3030G 9.5 9.5

3032 3032 ND ND 3032G 3032G 13.1 13.1

3033 3033 77.1 77.1

3033G 3033G 32.9 32.9

3036 3036 17.6 17.6

3036G 3036G 24.6 24.6

3041 3041 59.3 59.3

3041G 3041G 30.7 30.7

3042 3042 39.9 39.9

3042G 3042G 16.1 16.1

3043 3043 57.4 57.4

3043G 3043G 46.1 46.1

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[00161]

[00161]

Table 88 Table

VK3-20JYc1 Vk3-20Jk1 Common Common Light Light Chain Chain Antibodies Antibodies

Antibody Antibody %Blocking % Blocking of Antigen of In Solution Antigen EEIn Solution 2968 2968 68.9 68.9

2968G 2968G 15.2 15.2

2969 2969 10.1 10.1

2969G 2969G 23.6 23.6

2970 2970 34.3 34.3

2970G 2970G 41.3 41.3

2971 2971 6.3 6.3

2971 G 2971G 27.1 27.1

2972 2972 9.6 9.6

2972G 2972G 35.7 35.7

2973 2973 20.7 20.7

2973G 2973G 23.1 23.1

2974 2974 ND ND 2974G 2974G 22.0 22.0

2975 2975 8.7 8.7

2975G 2975G 19.2 19.2

2976 2976 4.6 4.6

2976G 2976G 26.7 26.7

Example 9. BIAcore Example 9. BAcoreTM AffinityDetermination Affinity Determinationfor forAntigen-Specific Antigen-Specific Common Common Light Light Chain Antibodies Chain Antibodies

[00162]

[00162] Equilibrium dissociation Equilibrium dissociation constants (KD) for constants (K) forselected selectedantibody antibody supernatants supernatants

were determined were determinedbybySPR SPR (Surface (Surface Plasmon Plasmon Resonance) Resonance) using ausing a BlAcore BIAcore TM T100 TMT instrument instrument

(GE Healthcare). (GE Healthcare). All All data was data obtained was using obtained HBS-EP using (10mM HBS-EP (10mMHepes, 150mM NaCl, Hepes,150mM NaCI, 0.3mM 0.3mM EDTA,0.05% EDTA, 0.05% Surfactant Surfactant P20, P20, pH pH 7.4)7.4) as both as both thethe running running andand sample sample buffers, buffers, at 25°C. at 25°C.

Antibodies were Antibodies werecaptured capturedfrom fromcrude crude supernatant supernatant samples samples on aon CM5 sensor CM5a sensor chip surface chip surface

previously derivatized previously derivatized with with aa high high density density of ofanti-human anti-human Fc Fc antibodies antibodies using using standard amine standard amine

coupling chemistry. During coupling chemistry. Duringthe thecapture capturestep, step,supernatants supernatantswere were injectedacross injected across theanti- the anti humanFcFc human surfaceatata aflow surface flowrate rateof of 33 µL/min, pL/min, for for aa total totalofof 3 minutes. 3 minutes.The The capture capture step step was was

followedbybyan an followed injection injection of of either either running running buffer buffer or analyte or analyte at a concentration at a concentration of 100 nMoffor 100 2 nM for 2 minutes at aa flow minutes at flow rate rate of of35 35 pL/min. Dissociation of µL/min. Dissociation of antigen antigen from from the the captured antibody was captured antibody was monitored for 66 minutes. monitored for minutes. The Thecaptured captured antibody antibody waswas removed removed by a by a brief brief injection injection of of 10 10 mM mM

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glycine, pH 1.5. All All sensorgrams 2023202781 04 May 2023

glycine, pH 1.5. weredouble sensorgrams were double referenced referenced by by subtracting subtracting sensorgrams sensorgrams from from

buffer buffer injections injectionsfrom fromthe theanalyte analytesensorgrams, thereby removing sensorgrams, thereby removingartifacts artifacts caused by caused by

dissociationofofthe dissociation theantibody antibody from from the capture the capture surface. surface. Binding Binding dataantibody data for each for eachwasantibody fit was fit to aa 1:1 to 1:1 binding binding model with mass model with transport using mass transport using BIAcore T100 BAcoreT100 Evaluation Evaluation software software v2.1. v2.1.

Results are shown Results are showninin Tables Tables9 9and and10. 10.

[00163]

[00163] The binding affinities The binding affinities ofofcommon light chain common light chain antibodies antibodies comprising the comprising the

rearrangementsshown rearrangements shown in in Tables Tables 3 and 3 and 4 vary, 4 vary, with with nearly nearly allallexhibiting exhibiting aa KD in the KD in the nanomolar range.TheThe nanomolar range. affinitydata affinity dataisis consistent consistent with with the the common lightchain common light chainantibodies antibodies resulting resulting from from the the combinatorial combinatorial association association of ofrearranged rearranged variable variable domains describedinin domains described

Tables 33 and Tables and 44 being beinghigh-affinity, high-affinity, clonally clonallyselected, and selected, andsomatically somaticallymutated. mutated. Coupled with Coupled with

data previously shown, data previously the common shown, the common lightchain light chainantibodies antibodiesdescribed described in in Tables Tables 3 and 3 and 4 4

comprise a collection comprise a collection of of diverse, diverse, high-affinity high-affinity antibodies antibodies that exhibit that exhibit specificity specificity fororone or for one

moreepitopes more epitopesononAntigen AntigenE.E.

[00164]

[00164]

Table Table 9 9

Vic1-39Jx5 VK1-39JK5 Common Light Chain Common Light Chain Antibodies Antibodies

AntigenE E 1OOnMAntigen 100nM Antibody Antibody KD (nM) KD (nM) T1/ (min) T/ (min) 2948 2948 8.83 8.83 28 28 2948G 2948G 95.0 95.0 1 1

2949 2949 3.57 3.57 18 18 2949G 2949G 6.37 6.37 9 9 2950 2950 4.91 4.91 17 17 2950G 2950G 13.6 13.6 5 5 2952 2952 6.25 6.25 7 7 2952G 2952G 7.16 7.16 44 2954 2954 2.37 2.37 24 24 2954G 2954G 5.30 5.30 9 9 2955 2955 14.4 14.4 6 6 2955G 2955G 12.0 12.0 44 2964 2964 14.8 14.8 6 6 2964G 2964G 13.0 13.0 9 9 2978 2978 1.91 1.91 49 49 2978G 2978G 1.80 1.80 58 58 2982 2982 6.41 6.41 19 19

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2982G 2982G 16.3 16.3 9 9

2985 2985 64.4 64.4 9 9

2985G 2985G 2.44 2.44 8 8

2987 2987 21.0 21.0 11 11

2987G 2987G 37.6 37.6 4 4 2996 2996 10.8 10.8 9 9

2996G 2996G 24.0 24.0 2 2

2023202781 2997 2997 7.75 7.75 19 19 2997G 2997G 151 151 1 1

3004 3004 46.5 46.5 14 14 3004G 3004G 1.93 1.93 91 91

3005 3005 2.35 2.35 108 108 3005G 3005G 6.96 6.96 27 27 3010 3010 4.13 4.13 26 26 3010G 3010G 2.10 2.10 49 49 3011 3011 59.1 59.1 55 3011G 3011G 41.7 41.7 55 3012 3012 9.71 9.71 20 20 3012G 3012G 89.9 89.9 2 2 3013 3013 20.2 20.2 20 20 3013G 3013G 13.2 13.2 4 4 3014 3014 213 213 4 4 3014G 3014G 36.8 36.8 33 3015 3015 29.1 29.1 11 11

3015G 3015G 65.9 65.9 00 3016 3016 4.99 4.99 17 17 3016G 3016G 18.9 18.9 4 4 3017 3017 9.83 9.83 88 3017G 3017G 55.4 55.4 22 3018 3018 11.3 11.3 36 36 3018G 3018G 32.5 32.5 33 3019 3019 1.54 1.54 59 59 3019G 3019G 2.29 2.29 42 42 3020 3020 5.41 5.41 39 39 3020G 3020G 41.9 41.9 66 3021 3021 50.1 50.1 66 3021G 3021G 26.8 26.8 4 4 3022 3022 25.7 25.7 17 17 3022G 3022G 20.8 20.8 12 12 3023 3023 263 263 9 9

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3023G 3023G 103 103 5 5 3024 3024 58.8 58.8 7 7 3024G 3024G 7.09 7.09 10 10 May 3025 3025 35.2 35.2 66 3025G 3025G 42.5 42.5 8 8 3027 3027 7.15 7.15 66 3027G 3027G 4.24 4.24 18 18 3028 3028 6.89 6.89 37 37 3028G 3028G 7.23 7.23 22 22 3030 3030 46.2 46.2 7 7 3030G 3030G 128 128 3 3 3032 3032 53.2 53.2 9 9 3032G 3032G 13.0 13.0 1 1 3033 3033 4.61 4.61 17 17 3033G 3033G 12.0 12.0 5 5 3036 3036 284 284 12 12 3036G 3036G 18.2 18.2 10 10 3041 3041 6.90 6.90 12 12 3041G 3041G 22.9 22.9 2 2 3042 3042 9.46 9.46 34 34 3042G 3042G 85.5 85.5 3 3 3043 3043 9.26 9.26 29 29 3043G 3043G 13.1 13.1 22 22

[00165]

[00165]

Table 10 Table 10

V-K3-20J-KI VK3-20JK1 Common Common Light Light Chain Chain Antibodies Antibodies

AntigenE E 100nMAntigen 100nM Antibody Antibody KD (nM) KD (nM) T (min) T/112(min) 2968 2968 5.50 5.50 8 8 2968G 2968G 305 305 0 0 2969 2969 34.9 34.9 2 2 2969G 2969G 181 181 1 1

2970G 2970G 12.3 12.3 3 3 2971G 2971G 32.8 32.8 22 22 2972 2972 6.02 6.02 13 13 2972G 2972G 74.6 74.6 26 26 2973 2973 5.35 5.35 39 39 2973G 2973G 11.0 11.0 44 44

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2974 2974 256 256 0 0 2974G 2974G 138 138 0 0 2975 2975 38.0 38.0 2 2 May 2975G 134 134 1 1 2975G 2976 2976 6.73 6.73 10 10 2976G 2976G 656 656 8 8

Example 10.Determination Example 10. Determination of Binding of Binding Specificities Specificities of Antigen-Specific of Antigen-Specific Common Common Light Light Chain Antibodies Chain AntibodiesbybyLuminexTM Assay Luminex Assay

[00166]

[00166] Selected anti-Antigen Selected anti-Antigen EE common common lightchain light chainantibodies antibodieswere were tested tested forfor their their

ability totobind ability to to bind thethe ECDECDofofAntigen AntigenEEand and Antigen Antigen E ECDvariants, E ECD variants,including including the the cynomolgous cynomolgous monkey monkey ortholog ortholog (Mf (Mf Antigen Antigen E), which E), which differs differs from from the the human human protein protein in in approximately 10% approximately 10%ofofits its amino aminoacid acidresidues; residues;a adeletion deletionmutant mutantofofAntigen AntigenE Elacking lackingthe thelast last 10 amino 10 aminoacids acidsfrom fromthe theC-terminal C-terminalend endofofthe theECD ECD (Antigen (Antigen E-ACT); E-ACT); and and two two mutants mutants

containingananalanine containing alanine substitution substitution at suspected at suspected locations locations of interaction of interaction withY with Ligand Ligand Y (Antigen E-Alal and (Antigen E-Ala1 andAntigenE-Ala2). AntigenE-Ala2).TheThe Antigen Antigen E proteins E proteins werewere produced produced in cells in CHO CHO cells and each and eachcontained containeda amyc-myc-His myc-myc-His C-terminal C-terminal tag.tag.

[00167]

[00167] For For the the binding binding studies, studies, Antigen E ECD Antigen E proteinororvariant ECD protein variant protein protein (described (described

above) from above) from 11 mL mLofofculture culture medium mediumwaswas captured captured by incubation by incubation for for 2 hr hr at 2 at room room

temperaturewith with 11 Xx 10 106microsphere microsphere (Luminex TM) beads covalently coated coated with anwith temperature (Luminex) beads covalently an anti-myc anti-myc

monoclonal antibody(MAb monoclonal antibody (MAb 9E10, 9E10, hybridoma hybridoma cell cell lineline CRL-1729TM; CRL-1729TM; ATCC,ATCC, Manassas, Manassas, VA). VA). The beads The beadswere were then then washed washed withwith PBS PBS before before use. use. Supernatants Supernatants containing containing anti-Antigen anti-Antigen E E common common light light chain chain antibodies antibodies were diluted were diluted 1:4 inand 1:4 in buffer buffer addedand added filter to 96-well to 96-well filter plates. A plates. A mock supernatantwith mock supernatant withnonoantibody antibodywas was used used as negative as negative control. control. The The beads beads containing containing the the

captured Antigen captured AntigenEEproteins proteinswere werethen thenadded added to to theantibody the antibody samples samples (3000 (3000 beads beads per well) per well)

and incubated and incubatedovernight overnightatat 4°C. Thefollowing 40C. The followingday, day,the thesample sample beads beads were were washed washed and and the the boundcommon bound common light light chain chain antibody antibody waswas detected detected withwith a R-phycoerythrin-conjugated a R-phycoerythrin-conjugated anti- anti

humanIgG human antibody.TheThe IgGantibody. fluorescence fluorescence intensity intensity of of the the beads beads (approximately (approximately 100 100 beads beads

countedfor counted for each eachantibody antibodysample samplebinding binding totoeach each Antigen Antigen E protein)waswas E protein) measured measured with with a a LuminexTM Luminex flowflow cytometry-based cytometry-based analyzer, analyzer, andmedian and the the median fluorescence fluorescence intensity intensity (MFI) (MFI) for for at least at least 100 100 counted beadsper counted beads perbead/antibody bead/antibody interactionwas interaction was recorded. recorded. Results Results are are shown shown

in Tables in 11 and Tables 11 and 12. 12.

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[00168]

[00168]

Table 11 Table 11 VKi1-39JK5 Common Vk1-39JK5 Common Light Light Chain Chain Antibodies Antibodies

MeanFluorescence Mean FluorescenceIntensity Intensity (MFI) (MFI) Antibody Antibody Antigen E- Antigen E- Antigen E- E- Antigen E- Antigen E- Antigen Antigen Antigen MfAntigenE ECD ACT Mf Antigen E ECD ACT Alal Ala1 E-Ala2 E-Ala2 2948 2948 1503 1503 2746 2746 4953 4953 3579 3579 1648 1648 2948G 2948G 537 537 662 662 2581 2581 2150 2150 863 863 2949 2949 3706 3706 4345 4345 8169 8169 5678 5678 5142 5142 2949G 2949G 3403 3403 3318 3318 7918 7918 5826 5826 5514 5514 2950 2950 3296 3296 4292 4292 7756 7756 5171 5171 4749 4749 2950G 2950G 2521 2521 2408 2408 7532 7532 5079 5079 3455 3455 2952 2952 3384 3384 1619 1619 1269 1269 168 168 911 911 2952G 2952G 3358 3358 1001 1001 108 108 55 55 244 244 2954 2954 2808 2808 3815 3815 7114 7114 5039 5039 3396 3396 2954G 2954G 2643 2643 2711 2711 7620 7620 5406 5406 3499 3499 2955 2955 1310 1310 2472 2472 4738 4738 3765 3765 1637 1637 2955G 2955G 1324 1324 1802 1802 4910 4910 3755 3755 1623 1623 2964 2964 5108 5108 1125 1125 4185 4185 346 346 44 44 2964G 2964G 4999 4999 729 729 4646 4646 534 534 91 91

2978 2978 6986 6986 2800 2800 14542 14542 10674 10674 8049 8049 2978G 2978G 5464 5464 3295 3295 11652 11652 8026 8026 6452 6452 2982 2982 4955 4955 2388 2388 13200 13200 9490 9490 6772 6772 2982G 2982G 3222 3222 2013 2013 8672 8672 6509 6509 4949 4949 2985 2985 1358 1358 832 832 4986 4986 3892 3892 1669 1669 2985G 2985G 43 43 43 43 128 128 244 244 116 116 2987 2987 3117 3117 1674 1674 7646 7646 5944 5944 2546 2546 2987G 2987G 3068 3068 1537 1537 9202 9202 6004 6004 4744 4744 2996 2996 4666 4666 1917 1917 12875 12875 9046 9046 6459 6459 2996G 2996G 2752 2752 1736 1736 8742 8742 6150 6150 4873 4873 2997 2997 5164 5164 2159 2159 12167 12167 8361 8361 5922 5922 2997G 2997G 658 658 356 356 3392 3392 2325 2325 1020 1020 3004 3004 2794 2794 1397 1397 8542 8542 6268 6268 3083 3083 3004G 3004G 2753 2753 1508 1508 8267 8267 5808 5808 4345 4345 3005 3005 5683 5683 2221 2221 12900 12900 9864 9864 5868 5868 3005G 3005G 4344 4344 2732 2732 10669 10669 7125 7125 5880 5880 3010 3010 4829 4829 1617 1617 2642 2642 3887 3887 44 44 3010G 3010G 3685 3685 1097 1097 2540 2540 3022 3022 51 51

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04 May 2023

3011 3011 2859 2859 2015 2015 7855 7855 5513 5513 3863 3863 3011G 3011G 2005 2005 1072 1072 6194 6194 4041 4041 3181 3181 3012 3012 3233 3233 2221 2221 8543 8543 5637 5637 3307 3307 3012G 3012G 968 968 378 378 3115 3115 2261 2261 1198 1198 3013 3013 2343 2343 1791 1791 6715 6715 4810 4810 2528 2528 3013G 3013G 327 327 144 144 1333 1333 1225 1225 370 370 3014 3014 1225 1225 1089 1089 5436 5436 3621 3621 1718 1718

2023202781 3014G 3014G 1585 1585 851 851 5178 5178 3705 3705 2411 2411 3015 3015 3202 3202 2068 2068 8262 8262 5554 5554 3796 3796 3015G 3015G 1243 1243 531 531 4246 4246 2643 2643 1611 1611 3016 3016 4220 4220 2543 2543 8920 8920 5999 5999 5666 5666 3016G 3016G 2519 2519 1277 1277 6344 6344 4288 4288 4091 4091 3017 3017 3545 3545 2553 2553 8700 8700 5547 5547 5098 5098 3017G 3017G 1972 1972 1081 1081 5763 5763 3825 3825 3038 3038 3018 3018 2339 2339 1971 1971 6140 6140 4515 4515 2293 2293 3018G 3018G 254 254 118 118 978 978 1020 1020 345 345 3019 3019 5235 5235 1882 1882 7108 7108 4249 4249 54 54 3019G 3019G 4090 4090 1270 1270 4769 4769 3474 3474 214 214 3020 3020 3883 3883 3107 3107 8591 8591 6602 6602 4420 4420 3020G 3020G 2165 2165 1209 1209 6489 6489 4295 4295 2912 2912 3021 3021 1961 1961 1472 1472 6872 6872 4641 4641 2742 2742 3021G 3021G 2091 2091 1005 1005 6430 6430 3988 3988 2935 2935 3022 3022 2418 2418 793 793 7523 7523 2679 2679 36 36 3022G 3022G 2189 2189 831 831 6182 6182 3051 3051 132 132 3023 3023 1692 1692 1411 1411 5788 5788 3898 3898 2054 2054 3023G 3023G 1770 1770 825 825 5702 5702 3677 3677 2648 2648 3024 3024 1819 1819 1467 1467 6179 6179 4557 4557 2450 2450 3024G 3024G 100 100 87 87 268 268 433 433 131 131

3025 3025 1853 1853 1233 1233 6413 6413 4337 4337 2581 2581 3025G 3025G 1782 1782 791 791 5773 5773 3871 3871 2717 2717 3027 3027 4131 4131 1018 1018 582 582 2510 2510 22 22 3027G 3027G 3492 3492 814 814 1933 1933 2596 2596 42 42 3028 3028 4361 4361 2545 2545 9884 9884 5639 5639 975 975 3028G 3028G 2835 2835 1398 1398 7124 7124 3885 3885 597 597 3030 3030 463 463 277 277 1266 1266 1130 1130 391 391 3030G 3030G 943 943 302 302 3420 3420 2570 2570 1186 1186 3032 3032 2083 2083 1496 1496 6594 6594 4402 4402 2405 2405 3032G 3032G 295 295 106 106 814 814 902 902 292 292 3033 3033 4409 4409 2774 2774 8971 8971 6331 6331 5825 5825 3033G 3033G 2499 2499 1234 1234 6745 6745 4174 4174 4210 4210

WO2011/097603 wo 2011/097603 PCT/US2011/023971 PCT/US2011/023971

2023202781 04 2023

3036 3036 1755 1755 1362 1362 6137 6137 4041 4041 1987 1987 3036G 3036G 2313 2313 1073 1073 6387 6387 4243 4243 3173 3173 3041 3041 3674 3674 2655 2655 8629 8629 5837 5837 4082 4082 May 3041G 3041G 2519 2519 1265 1265 6468 6468 4274 4274 3320 3320 3042 3042 2653 2653 2137 2137 7277 7277 5124 5124 3325 3325 3042G 3042G 1117 1117 463 463 4205 4205 2762 2762 1519 1519 3043 3043 3036 3036 2128 2128 7607 7607 5532 5532 3366 3366 3043G 3043G 2293 2293 1319 1319 6573 6573 4403 4403 3228 3228

[00169]

[00169]

12 Table 12 Table

V3-20Jk1 Common V3-20J1c Common LightChain Light Antibodies ChainAntibodies Intensity (MFI) FluorescenceIntensity Mean Fluorescence Mean (MFI) Antibody Antibody Antigen E- Antigen E- Antigen E- Antigen E- Antigen E- Antigen E- Antigen E- Antigen E- MfAntigenE Ala2 Mf Antigen E ECD ECD ACT ACT Alai Ala1 Ala2 2968 2968 6559 6559 3454 3454 14662 14662 3388 3388 29 29 2968G 2968G 2149 2149 375 375 9109 9109 129 129 22 22 2969 2969 2014 2014 1857 1857 7509 7509 5671 5671 3021 3021 2969G 2969G 1347 1347 610 610 6133 6133 4942 4942 2513 2513 2970 2970 5518 5518 1324 1324 14214 14214 607 607 32 32 2970G 2970G 4683 4683 599 599 12321 12321 506 506 31 31

2971 2971 501 501 490 490 2506 2506 2017 2017 754 754 2971G 2971G 578 578 265 265 2457 2457 2062 2062 724 724 2972 2972 2164 2164 2158 2158 8408 8408 6409 6409 3166 3166 2972G 2972G 1730 1730 992 992 6364 6364 4602 4602 2146 2146 2973 2973 3527 3527 1148 1148 3967 3967 44 44 84 84 2973G 2973G 1294 1294 276 276 1603 1603 28 28 44 44 2974 2974 1766 1766 722 722 8821 8821 241 241 19 19 2974G 2974G 2036 2036 228 228 8172 8172 135 135 26 26 2975 2975 1990 1990 1476 1476 8669 8669 6134 6134 2468 2468 2975G 2975G 890 890 315 315 4194 4194 3987 3987 1376 1376 2976 2976 147 147 140 140 996 996 1079 1079 181 181

2976G 2976G 1365 1365 460 460 6024 6024 3929 3929 1625 1625

[00170]

[00170] The anti-Antigen EE common The anti-Antigen common light light chainantibody chain antibody supernatants supernatants exhibited exhibited high high

specific binding specific binding to tothe thebeads beads linked linked to toAntigen Antigen E-ECD. Forthese E-ECD. For thesebeads, beads,thethenegative negative control mock control supernatantresulted mock supernatant resultedinin negligible negligible signal signal (<10 (<10 MFI) MFI) when combined when combined with with thethe

Antigen E-ECD Antigen E-ECDbead bead sample, sample, whereas whereas the supernatants the supernatants containing containing anti-Antigen anti-Antigen E common E common

55

WO2011/097603 wo 2011/097603 PCT/US2011/023971 PCT/US2011/023971

light chain antibodiesexhibited exhibited strong binding signalsignal (average MFIfor of 98 2627 for 98 antibody 2023202781 04 May 2023

light chain antibodies strong binding (average MFI of 2627 antibody

MFI>>500 supernatants; MFI supernatants; for91/98 500for 91/98antibody antibodysamples). samples).

[00171]

[00171] As measureofofthe As aa measure theability ability of of the theselected selected anti-Antigen anti-Antigen EE common light chain common light chain antibodiestotoidentify antibodies identifydifferent differentepitopes epitopes on the on the ECD ECD of of Antigen Antigen E, the relative E, the relative binding binding of the of the antibodies to antibodies to the the variants variants were were determined. All four determined. All four Antigen variants were Antigen EE variants capturedtoto the were captured the anti-myc Luminex anti-myc LuminexTM beadsasasdescribed TM beads described above above for for thethe native native Antigen Antigen E-ECD E-ECD binding binding

studies, and studies, andthetherelative relative binding binding ratios ratios (MFIvariant/MFlAntigenE-ECD) (MFlvariant/MFIAntigen were E-ECD)were determined. determined. For 98 For 98 tested common tested lightchain common light chainantibody antibodysupernatants supernatants shown shown in Tables in Tables 11 and 11 and 12, average 12, the the average ratios E-ECD)differed (MFlvariant/MFAntigen E-ECD) ratios (MFIvariant/MFIAntigen foreach differedfor variant, likely each variant, likely reflecting reflecting different different capture capture amounts amounts of of proteins proteins on beads on the the beads (average (average ratios ofratios 0.61, of 0.61, 2.9, 2.0, 2.9, 2.0,for and 1.0 and 1.0 for Antigen E- Antigen E

ACT,Antigen CT, Antigen E-Alal, E-Ala1, Antigen Antigen E-Ala2, E-Ala2, andand Mf Antigen Mf Antigen E, respectively). E, respectively). For For eacheach protein protein

variant, the variant, the binding bindingfor fora asubset subsetofof thethe9898tested common tested light chain common light chain antibodies antibodies showed showed

greatly reduced greatly reduced binding, binding, indicating indicating sensitivity sensitivity to mutation to the the mutation that characterized that characterized a given a given variant. For variant. For example, 19ofofthe example, 19 the common common lightchain light chainantibody antibody samples samples bound bound to the to the Mf Mf AntigenE Ewith Antigen with MFivariant/MFIAntigenE-ECD MFIvariant/MFIAntigen of Since <8%. E-ECDof <8%. many many Since in this in thisinclude group group high include or high or moderately moderately high high affinity affinity antibodies antibodies (5 with (5 with 5nM, 5nM,< 15 KD < KD with 15 KD with KD <it50isnM), < 50 nM), it is likely likely that the that the lowersignal lower signalfor forthis thisgroup group results results from from sensitivity sensitivity to the to the sequence sequence (epitope) (epitope) differences differences

betweennative between nativeAntigen AntigenE-ECD E-ECDand and a given a given variant variant rather rather than than from from lower lower affinities. affinities.

[00172]

[00172] These dataestablish These data establish that that the the common lightchain common light chainantibodies antibodiesdescribed describedinin

Tables and44indeed Tables 33 and indeedrepresent diversegroup representa adiverse group of of Antigen-E-specificcommon Antigen-E-specific common lightlight chain chain

antibodies that antibodies that specifically specificallyrecognize recognizemore more than than one epitope on one epitope on Antigen AntigenE.E.

56

THE CLAIMS DEFINING THE CLAIMS DEFINING THE THE INVENTION INVENTION ARE ARE AS AS FOLLOWS: FOLLOWS:

1. 1. A targeting A targeting vector vector comprising: comprising:

(i) (i) aa 5' 5' homology armcomprising homology arm comprising a nucleotidesequence a nucleotide sequence corresponding corresponding to ato a

genomic target sequence genomic target sequencethat thatis is upstream upstreamto to aa 5'-most 5'-most mouse mouse gene VK gene segment segment in an in an

unmodified endogenous unmodified endogenous mouse mouse kappa kappa lightlight chain chain locus; locus;

(ii) (ii) aa selection marker; selection marker;

(iii) (iii)a a promotersequence, VKpromoter sequence, (iv) (iv) aasingle singlerearranged rearrangedimmunoglobulin light chain immunoglobulin light chain /VK/JK sequence; sequence; and and (v) (v) aa 3' 3'homology armcomprising homology arm comprising a nucleotidesequence a nucleotide sequence corresponding corresponding to ato a

genomic target sequence genomic target sequencethat thatis is downstream downstreamofof anan endogenous endogenous mouse mouse JK5 gene JK5 gene segment segment in in an unmodified endogenous unmodified endogenous mouse mouse kappa kappa lightlight chain chain locus, locus, wherein wherein the the 3' homology 3' homology arm arm

comprises comprises aa mouse mouseCKCK exon. exon.

2. 2. The targeting The targeting vector of of claim 1, wherein claim 1, the single wherein the single rearranged rearranged immunoglobulin light immunoglobulin light

chain chain VK/JK sequence / sequence comprises comprises a human a human VK1-39 Vk1-39 or a a human or human VK3-20 Vk3-20 sequence. sequence.

3. 3. Thetargeting The targetingvector vector of claim of claim 1 or 1claim or claim 2, wherein 2, wherein the rearranged the single single rearranged light light chain chain VK/JK sequence / sequence is ais human a human VK1-39/JK5 Vk1-39/Jk5 sequence. sequence.

4. 4. Thetargeting The targetingvector vector of claim of claim 1 or 1claim or claim 2, wherein 2, wherein the rearranged the single single rearranged light light chain chain VK/JK / sequence sequence is ais human a human VK3-20/JK1 Vk3-20/Jk1 sequence. sequence.

5. 5. The targeting The targeting vector of of any any one of claims 1-4, one of 1-4, wherein the wherein the VK promoter promoter sequence sequence is is

a human VK3-15 human Vk3-15 promoter promoter sequence. sequence.

6. 6. Thetargeting The targetingvector vector of any of any one one of claims of claims 1-5, wherein 1-5, wherein the targeting the targeting vector vector further further comprises comprises aa mouse mouse VK3-7 leadersequence. 3-7 leader sequence.

57

7. 7. A host A host cell cell comprising: comprising:

(a) (a) aa first firstnucleic nucleicacid sequence acid sequencethat thatencodes encodesafirst immunoglobulin a first immunoglobulin heavy heavy

chain, chain, the the first firstnucleic acid nucleic sequence acid sequencecomprising comprising aafirst firsthuman human immunoglobulin heavy immunoglobulin heavy

chain variableregion chain variable region sequence sequence that that was identified was identified from a from B cell aofB acell ofmouse, first a firstwherein mouse, wherein the first the first mouse has mouse has been been immunized immunized with a with first a first antigen antigen of interest of interest includingincluding a first a first epitope, epitope, wherein the wherein the first first human immunoglobulin human immunoglobulin heavy heavy chain chain variable variable region region sequence sequence encodes encodes a a first firsthuman immunoglobulin human immunoglobulin heavy heavy chain chain variable variable domain domain thatthat recognizes recognizes the the first first epitope, epitope,

and whereinthe and wherein the first first mouse comprisesininits mouse comprises its germline genome: germline genome:

(i) (i) an anengineered engineered mouse immunoglobulin mouse immunoglobulin kappa kappa light light chain chain locus locus

comprising: comprising: aa VK promoter promoter sequence, sequence, a single a single rearranged rearranged immunoglobulin immunoglobulin light light

chain chain VK/JK sequence, / sequence, andand a mouse a mouse CK CK sequence,and sequence, and (ii) (ii)an anengineered engineered mouse immunoglobulin mouse immunoglobulin heavy heavy chain chain locus locus comprising: comprising:

unrearranged human unrearranged human immunoglobulin immunoglobulin heavyheavy chain chain variable variable region region gene gene segments segments

operably linked to operably linked to a mouse immunoglobulin mouse immunoglobulin heavy heavy chain chain constant constant region region sequence sequence

at at the the endogenous heavychain endogenous heavy chainloci; loci; and and (b) (b) a second a nucleic acid second nucleic acid sequence sequence that that encodes encodes an animmunoglobulin immunoglobulin light light chain chain

comprising animmunoglobulin comprising an immunoglobulin kappa kappa light light chain chain variable variable region region sequence sequence comprising comprising the the

single single rearranged rearranged immunoglobulin lightchain immunoglobulin light chain/ VK/JK sequence, sequence, or a somatically or a somatically

hypermutatedversion hypermutated versionthereof. thereof.

8. 8. The host The host cell cell of of claim claim 7, 7,wherein wherein the the single singlerearranged rearranged immunoglobulin light chain immunoglobulin light chain VK/JK / sequence sequence comprises comprises a human a human VK1-39 Vk1-39 a human or aorhuman 3-20VK3-20 sequence. sequence.

9. 9. host cell The host The cell of of claim claim 77 or or claim claim 8, wherein the 8,wherein the single singlerearranged rearranged light lightchain chainVK/JK /

sequence is aa human sequence is VK1-39/JK5 human Vk1-39/Jk5 sequence. sequence.

10. 10. The host The host cell cell of of claim claim 77 or or claim claim 8, 8,wherein wherein the the single singlerearranged rearranged light lightchain chainVK/JK /

sequence is aa human sequence is V3-20/JK1 human Vk3-20/Jk1 sequence. sequence.

58

11. 11. The host The host cell cell of of any any one one of claims claims 7-10, 7-10, wherein the wherein the VK promoter promoter sequence sequence is a is a

human Vk3-15 human VK3-15promoter promoter sequence. sequence.

12. 12. The The hosthost cellcell of of anyany oneone of claims of claims 7-11, 7-11, wherein wherein the the engineered engineered mouse mouse

immunoglobulin kappa immunoglobulin kappa light light chain chain locus locus furthercomprises further comprises a mouse a mouse V3-7 Vk3-7 leader leader sequence. sequence.

13. 13. Thehost The hostcell cellofofany any oneone of claims of claims 7-12,7-12, further further comprising comprising a third acid a third nucleic nucleic acid sequence that encodes sequence that encodes aa second secondimmunoglobulin immunoglobulin heavy heavy chain chain comprising comprising a second a second human human

immunoglobulin heavy immunoglobulin heavy chain chain variable variable region region sequence sequence thatthat waswas identified identified from from a Ba cell B cell of of a a

second mouse,wherein second mouse, wherein thesecond the second mouse mouse has has beenbeen immunized immunized with awith a second second antigen antigen of of interest interest including including aasecond second epitope, epitope,wherein wherein the the second second human immunoglobulin human immunoglobulin heavy heavy

chain variable variable region sequence encodesa asecond sequence encodes secondhuman human immunoglobulin immunoglobulin heavy heavy chain chain

variable domain that recognizes domain that recognizes the the second second epitope, epitope, and and wherein whereinthe thesecond secondmouse mouse comprises in its comprises in its germline genome: germline genome:

(i) (i) an engineered mouse an engineered mouseimmunoglobulin immunoglobulin kappa kappa lightlight chain chain locus locus comprising: comprising: a a VK promoter promoter sequence,the sequence, the single single rearranged rearrangedimmunoglobulin immunoglobulinlight chain light VK/JK chain sequence, / sequence,

and and aa mouse mouseCKCKsequence, sequence,andand (ii) an an (ii) engineered engineered mouse mouse immunoglobulin immunoglobulin heavylocus heavy chain chaincomprising: locus comprising: unrearranged human unrearranged human immunoglobulin immunoglobulin heavyheavy chain chain variable variable region region gene gene segments segments operably operably

linked to to aa mouse immunoglobulin mouse immunoglobulin heavy heavy chain chain constant constant region region sequence sequence at the at the endogenous endogenous

heavy chain heavy chain loci; loci; and and

wherein the wherein the first first and and the thesecond second immunoglobulin heavy immunoglobulin heavy chains chains pair pair with with the the

immunoglobulin lightchain immunoglobulin light chainencoded encoded by by thethe second second nucleic nucleic acid acid sequence. sequence.

14. A method 14. A method forfor making making an an antibodycomprising: antibody comprising: expressing expressing in in a a single single cell: cell:

(a) (a) aa first firstnucleic nucleicacid sequence acid sequencethat thatencodes encodesa afirst immunoglobulin first immunoglobulin heavy heavy

chain, chain, the the first firstnucleic acid nucleic sequence acid sequencecomprising comprising aafirst firsthuman human immunoglobulin heavy immunoglobulin heavy

chain variableregion chain variable region sequence sequence that that was identified was identified from a from B cellaof B acell ofmouse, first a firstand mouse, and

59

Claims (27)

1. wherein the first mouse has been immunized with a first antigen of interest including a first wherein the first mouse has been immunized with a first antigen of interest including a first
2. epitope, epitope, wherein the first wherein the first human immunoglobulin human immunoglobulin heavy heavy chain chain variable variable region region sequence sequence
3. encodes encodes aa first first human immunoglobulin human immunoglobulin heavy heavy chain chain variable variable domain domain that that recognizes recognizes the the
4. first first epitope, andwherein epitope, and whereinthethe first first mouse mouse comprises comprises in its in its germline germline genome: genome:
5. (i) (i) an anengineered engineered mouse immunoglobulin mouse immunoglobulin kappa kappa light light chain chain locus locus
6. comprising: comprising: aa VK promoter promoter sequence, sequence, a single a single rearranged rearranged immunoglobulin immunoglobulin light light
7. chain Vk/Jk chain VK/JKsequence, sequence,and anda amouse mouseCK CK sequence, sequence, and and
8. (ii) (ii)an anengineered engineered mouse immunoglobulin mouse immunoglobulin heavy heavy chain chain locus locus comprising: comprising:
9. unrearranged human unrearranged human immunoglobulin immunoglobulin heavyheavy chain chain variable variable region region gene gene segments segments
10. operably linked to operably linked to aa mouse immunoglobulin mouse immunoglobulin heavy heavy chain chain constant constant region region sequence sequence
11. at at the the endogenous heavychain endogenous heavy chainloci; loci; and and (b) (b) aa second nucleic acid second nucleic acid sequence sequence that that encodes encodes ananimmunoglobulin immunoglobulin light light chain, chain,
12. the second the nucleic acid second nucleic acid sequence comprisingananimmunoglobulin sequence comprising immunoglobulin kappa kappa lightlight chain chain variable variable
13. region sequence region sequence comprising comprisingthethesingle singlerearranged rearrangedimmunoglobulin immunoglobulin light light chain chain / VK/JK
14. sequence, or aa somatically sequence, or hypermutatedversion somatically hypermutated versionthereof; thereof; maintaining maintaining thethe cellunder cell under conditions conditions sufficient sufficient to express to express the antibody; the antibody; and and isolating theantibody. isolating the antibody.
15. 15. 15. The method The methodofofclaim claim14,14,wherein whereinthethesingle singlerearranged rearrangedimmunoglobulin immunoglobulin light light chain chain

    VK/JK / sequence sequence comprises comprises a human a human VK1-39 Vk1-39 a human or aorhuman VK3-20 Vk3-20 sequence. sequence.
16. 16. 16. The The method method of claim of claim 14 or 14 or claim claim 15, wherein 15, wherein the single the single rearranged rearranged lightlight chainchain

    VK/JK / sequence sequence is ais human a human VK1-39/JK5 Vk1-39/Jk5 sequence. sequence.
17. 17. 17. The The method method of claim of claim 14 or 14 or claim claim 15, wherein 15, wherein the single the single rearranged rearranged lightlight chainchain

    sequenceisis aa human VK/JK sequence Vk/Jk humanVk3-20/Jk1 VK3-20/JK1 sequence. sequence.
18. 18. 18. The The method method ofone of any anyofone of claims claims 14-17,14-17, wherein wherein the VK promoter the promoter sequence sequence is a is a humanVk3-15 human VK3-15promoter promoter sequence. sequence.

    60
19. 19. 19. The The method method ofone of any anyofone of claims claims 14-18,14-18, wherein wherein the engineered the engineered mouse mouse immunoglobulin kappa immunoglobulin kappa light light chain chain locus locus furthercomprises further comprises a mouse a mouse V3-7 Vk3-7 leader leader sequence. sequence.
20. 20. The The 20. method method of anyofone anyofone of claims claims 14-19,14-19, wherein wherein thecomprises the cell cell comprises a third a third nucleic nucleic

    acid sequence that encodes sequence that encodes aa second secondimmunoglobulin immunoglobulin heavy heavy chain chain comprising comprising a second a second

    humanimmunoglobulin human immunoglobulin heavy heavy chainchain variable variable region region sequence sequence that that was identified was identified from from a B aB cell cell of of aasecond second mouse, and wherein mouse, and whereinthe the second secondmouse mousehashas been been immunized immunized with with a second a second

    antigen of of interest interestincluding includinga asecond second epitope, epitope,wherein wherein the thesecond second immunoglobulin heavy immunoglobulin heavy

    chain variable variable region sequence encodesa asecond sequence encodes secondhuman human immunoglobulin immunoglobulin heavy heavy chain chain

    variable variable domain that recognizes domain that recognizes the the second second epitope, epitope, and and wherein whereinthe thesecond secondmouse mouse comprises in its comprises in its germline genome: germline genome:

    (i) (i) an engineered engineered mouse mouseimmunoglobulin immunoglobulin kappa kappa lightlight chain chain locus locus comprising: comprising: a a VK promoter promoter sequence,the sequence, the single single rearranged rearrangedimmunoglobulin immunoglobulinlight chain light VK/JK chain sequence, / sequence,

    and and aa mouse mouseCKCKsequence, sequence,andand (ii) (ii) an an engineered engineered mouse mouse immunoglobulin immunoglobulin heavylocus heavy chain chaincomprising: locus comprising: unrearranged human unrearranged human immunoglobulin immunoglobulin heavyheavy chain chain variable variable region region gene gene segments segments operably operably

    linked to to aa mouse immunoglobulin mouse immunoglobulin heavy heavy chain chain constant constant region region sequence sequence at the at the endogenous endogenous

    heavy chain heavy chain loci, loci, and and

    wherein the wherein the first first and and the thesecond second immunoglobulin heavy immunoglobulin heavy chains chains pair pair with with the the

    immunoglobulin lightchain immunoglobulin light chainencoded encoded by by thethe second second nucleic nucleic acid acid sequence. sequence.
21. 21. The The 21. method method of claim of claim 14, wherein 14, wherein the first the first and and the the second second immunoglobulin immunoglobulin heavy heavy chains are are fully fullyhuman immunoglobulin human immunoglobulin heavy heavy chains chains and and the the immunoglobulin immunoglobulin light light chainchain

    is is aa fully fullyhuman human immunoglobulin lightchain. immunoglobulin light chain.
22. 22. A mouse 22. A mouse embryonic embryonic stemcell stem (ES) (ES)that cellhas thatbeen has genetically been genetically modified modified soitthat so that it comprises in its comprises in its genome: genome:

    61

    (a) (a) an engineered engineered mouse mouseimmunoglobulin immunoglobulin kappa kappa lightlight chain chain locus locus comprising: comprising: a a VK promoter promoter sequence, sequence, a single a single rearranged rearranged VK/JK Vk/Jk sequence; sequence; and aand a mouse mouse CK sequence; CK sequence; and and (b) (b) an engineered mouse an engineered mouseimmunoglobulin immunoglobulin heavy heavy chainchain locuslocus comprising: comprising: a a plurality of plurality ofhuman immunoglobulin human immunoglobulin heavy heavy chain chain variable variable region region gene gene segments segments operably operably

    linked to to an an endogenous mouse endogenous mouse immunoglobulin immunoglobulin heavyheavy chainchain constant constant region, region, wherein wherein the the

    plurality of plurality ofhuman immunoglobulin human immunoglobulin heavy heavy chain chain variable variable region region gene gene segments segments replaces replaces

    endogenous mouse endogenous mouse immunoglobulin immunoglobulin heavyheavy chain chain variable variable regionregion gene segments gene segments at the at the

    endogenous mouse endogenous mouse immunoglobulin immunoglobulin heavy heavy chain chain locus.locus.
23. 23. The The 23. ES cell ES cell of claim 22, 22, of claim wherein wherein the single the single rearranged rearranged V/JK sequence / sequence is a is a single single rearranged humanVk1-39/Jk rearranged human VK1-39/JK sequence sequence or aorsingle a single rearranged rearranged human human V3-20/JK V3-20/J sequence. sequence.
24. 24. The The 24. ES cell ES cell of claim of claim 22claim 22 or 23, 23, or claim wherein wherein the single the single rearranged rearranged light light chain chain / VK/JK

    sequence is aa human sequence is VK1-39/JK5 human Vk1-39/Jk5 sequence. sequence.
25. 25. The The 25. ES cell ES cell of claim of claim 22claim 22 or 23, 23, or claim wherein wherein the single the single rearranged rearranged light light chain chain / VK/JK

    sequence is aa human sequence is V3-20/JK1 human Vk3-20/Jk1 sequence. sequence.
26. 26. The The 26. ES cell ES cell of any of any one one of claims of claims 22-25, 22-25, wherein wherein the the VK promoter promoter sequencesequence is a is a human Vk3-15 human VK3-15promoter promoter sequence. sequence.
27. 27. The The 27. ES cell ES cell of any of any one one of claims of claims 22-26, 22-26, wherein wherein the engineered the engineered mousemouse

    immunoglobulin kappa immunoglobulin kappa light light chain chain locus locus furthercomprises further comprises a mouse a mouse V3-7 Vk3-7 leader leader sequence. sequence.

    62

    2023202781 04 May 2023 Mouse CK Deleted Endogenous Mouse IgK Locus 

    Mouse enh enh

    C

    Deleted Endogenous

    Mouse IgK Locus Mouse Mouse Rearranged Germline VK3-7 Leader CK

    enh

    enh Human VK1-39JK5 FRT NEO FRT Targeting Vector Human Human enh enh VK3-15 VK1-39JK5 Promoter 1/3

    Mouse

    Mouse ³-7 Leader

    Rearranged Germline C + FLP

    Human VK1-39JK5 FRT FRT

    NEO

    Targeting Vector Human

    Human enh enh

    ³-15 VK1-39JK5 Mouse Mouse

    Promoter VK3-7 Leader CK Engineered Human VK1- FRT 1/3

    39JK5 Locus Human Human enh enh VK3-15 VK1-39JK5

    + FLP Promoter

    Figure 1 Mouse

    Mouse

    2023202781 04 May 2023 Mouse CK Deleted Endogenous Mouse IgK Locus 

    Mouse enh enh

    C

    Deleted Endogenous

    Mouse IgK Locus Mouse Mouse Rearranged Germline VK3-7 Leader CK

    enh

    enh Human VK3-20JK1 FRT NEO FRT Targeting Vector Human Human enh enh VK3-15 VK3-20JK1 Promoter 2/3

    Mouse

    Mouse ³-7 Leader

    Rearranged Germline C + FLP

    FRT FRT

    NEO

    Human V3-20J1 Targeting Vector Human

    Human enh enh

    ³-15 V3-20J1 Mouse Mouse

    Promoter VK3-7 Leader CK Engineered Human VK3- FRT 2/3

    20JK1 Locus Human Human enh enh VK3-15 VK3-20JK1

    + FLP Promoter

    Figure 2 Mouse

    Mouse

    2023202781 04 May 2023 Mouse CK Deleted Endogenous Mouse IgK Locus 

    Mouse enh enh

    C

    Deleted Endogenous

    Mouse IgK Locus Mouse Mouse Rearranged Germline VK3-7 Leader CK

    enh

    enh Human VpreBJl5 FRT NEO FRT Targeting Vector Human Human enh enh VK3-15 VpreBJl5 Promoter 3/3

    Mouse

    Mouse 3-7 Leader

    Rearranged Germline C + FLP

    FRT FRT

    NEO

    Human VpreBJX5 Targeting Vector Human

    Human enh enh

    ³-15 VpreBJX5 Mouse Mouse

    Promoter VK3-7 Leader CK Engineered Human FRT 3/3

    VpreBJl5 Locus Human Human enh enh VK3-15 VpreBJl5

    + FLP Promoter

    Figure 3 Mouse

    Mouse