JP2018502550A - Modified antigen-binding polypeptide constructs and uses thereof - Google Patents

Abstract

A heterodimer that can comprise a first heterodimer and a second heterodimer, wherein each heterodimer comprises an immunoglobulin heavy chain or fragment thereof and an immunoglobulin light chain or fragment thereof The present invention provides a pair. At least one of the heterodimers can include one or more amino acid modifications in the CH1 and / or CL domains, one or more amino acid modifications in the VH and / or VL domains, or combinations thereof. . The modified amino acid can be part of the light chain and heavy chain interface, and the first heterodimer when the two heavy chains and the two light chains of the heterodimer pair are co-expressed in the cell. Are typically modified so that the heavy chain of each pair is preferentially paired with one light chain but not the other, giving a preferential pairing between each heavy chain and the desired light chain. produce. Similarly, the heavy chain of the second heterodimer typically preferentially pairs with the second light chain rather than the first.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a priority of US Provisional Application No. 62 / 003,663 filed May 28, 2014 and US Provisional Patent Application No. 62 / 154,055 filed April 28, 2015. Claiming the benefit of rights, these applications are hereby incorporated by reference in their entirety for all purposes.

  This application is based on PCT / CA2013 / 050914 filed on November 28, 2013, US provisional application 61 / 730,906 filed on November 28, 2012, and US patent provisional application filed on February 6, 2013. 61 / 761,641, US provisional application 61 / 818,874 filed May 2, 2013 and US provisional application 61 / 869,200 filed August 23, 2013. , The entire disclosure of each is hereby incorporated by reference in its entirety for all purposes.

SEQUENCE LISTING This application contains a Sequence Listing that is provided electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on May 29, 2015, and is 97993-945204 (000110PC) _SL. It is called txt and has a size of 27,012 bytes.

  Bispecific antibodies can bind to two different epitopes. These epitopes can be on the same antigen, or each epitope can be on a different antigen. This bispecific antibody feature makes it an attractive tool for a variety of therapies and has the therapeutic benefit of targeting or mobilizing more than one molecule in the treatment of disease. One approach to forming bispecific antibodies involves the co-expression of two unique antibody heavy chains and two unique antibody light chains. As the antibody heavy chain proceeds to bind relatively irregularly with the antibody light chain, it is still a challenge to accurately form bispecific antibodies in a format resembling wild-type. As a result of this irregular pairing, the co-expression of two antibody heavy chains and two antibody light chains naturally results in an irregular heavy chain-light chain pairing. This mispairing is still a major challenge for the generation of bispecific therapeutics, and a homogenous pairing is an essential requirement for good manufacturability and biological efficacy.

  Several approaches have been described for preparing bispecific antibodies in which a particular antibody light chain or fragment is paired with an antibody heavy chain or fragment. A review of various approaches to address this problem can be found in Klein et al. (2012) mAbs 4: 6, 1-11 (Non-Patent Document 1). International patent application PCT / EP2011 / 056388 (International Application Publication No. 2011/131746), which is incubated under reducing conditions, shows a “Fab arm between two monospecific IgG4 or IgG4-like antibodies. Describes an in vitro method for generating heterodimeric proteins that introduces asymmetric mutations into the CH3 regions of two monospecific starting proteins in order to direct an exchange with a "or" half molecule "orientation .

  Schaefer et al. (Roche Diagnostics GmbH) assembles two heavy and two light chains derived from two existing antibodies into a human bivalent bispecific IgG antibody without using an artificial linker. A method is described (PNAS (2011) 108 (27): 11187-11192 (Non-patent Document 2)). This method involves exchanging the heavy and light chain domains within half of the antigen-binding fragment (Fab) of the bispecific antibody.

  Strop et al. (Rinat-Pfizer Inc.) describes a method for generating stable bispecific antibodies by separately expressing and purifying two antibodies of interest and then mixing together under certain redox conditions. (J. Mol. Biol. (2012) 420: 204-19 (non-patent document 3)).

  Zhu et al. (Genentech) have genetically engineered mutations in the VL / VH interface of a diabody construct consisting of variable domain antibody fragments that are completely devoid of constant domains to generate a heterodimeric diabody. (Protein Science (1997) 6: 781-788 (Non-patent Document 4)). Similarly, Igawa et al. (Chugai) also engineered mutations in the VL / VH interface of single-chain diabody to promote selective expression of diabody and inhibit conformational isomerism (Protein Engineering, Design & Selection (2010) 23: 667-677 (Non-Patent Document 5)).

  US Patent Publication No. 2009/0182127 (Novo Nordisk, Inc.) modifies amino acid residues at the Fc and CH1: CL interfaces of the light chain-heavy chain pair, The generation of bispecific antibodies is described by reducing the ability of a chain to interact with the other pair of heavy chains.

  US Patent Publication No. 2014/0370020 (Chugai) describes the association of CH1 and CL regions of antibodies by replacing amino acids present at the interface of these regions with charged amino acids. To do.

International Patent Application PCT / EP2011 / 056388 (International Application Publication No. 2011/131746) US Patent Publication No. 2009/0182127 US Patent Publication No. 2014/0370020

Klein et al. (2012) mAbs 4: 6, 1-11 PNAS (2011) 108 (27): 11187-11192 J. et al. Mol. Biol. (2012) 420: 204-19 Protein Science (1997) 6: 781-788 Protein Engineering, Design & Selection (2010) 23: 667-677

At least a first heterodimer and a second heterodimer, wherein the first heterodimer comprises a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain poly A peptide sequence (L1), wherein the second heterodimer comprises a second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin light chain polypeptide sequence (L2); At least one of the H1 or L1 sequences of the heterodimer is different from the corresponding H2 or L2 of the second heterodimer, and H1 and H2, respectively, are at least a heavy chain variable domain ( _VH domain) and a heavy A chain constant domain (C _H1 domain), wherein L1 and L2 each contain at least a light chain variable domain ( _VL domain) and a light chain constant domain (C _L domain), and H1, H 2, at least one of L1 and L2 comprises at least one amino acid modification in at least one constant domain and / or at least one variable domain, H1 preferentially pairs with L1 over L2, and H2 Described herein are isolated antigen-binding polypeptide constructs that preferentially pair with L2 over L1.

In some embodiments, the construct further comprises a heterodimeric Fc, wherein the Fc comprises at least two C _H3 sequences, and the Fc comprises the first with or without one or more linkers. Bound to the heterodimer and the second heterodimer, the dimerized C _H3 sequence has a melting temperature (Tm) of about 68 ° C. or higher as measured by differential scanning calorimetry (DSC). The construct is bispecific.

  In some embodiments, the at least one amino acid modification is selected from the at least one amino acid modification shown in the tables or examples.

  In some embodiments, when H1, H2, L1 and L2 are coexpressed in a cell or mammalian cell, or when H1, H2, L1 and L2 are coexpressed in a cell free expression system, or H1, H2, L1 and When L2 is co-produced, or when H1, H2, L1 and L2 are co-produced via a redox production method, H1 preferentially pairs with L1 over L2, and H2 is over L1. Also preferentially pair with L2.

In some embodiments, at least one of H1, H2, L1, and L2 is such that H1 preferentially pairs with L1 over L2 and / or H2 preferentially pairs with L2 over L1. As such, it comprises at least one amino acid modification of the V _H and / or _VL domain and at least one amino acid modification of the C _H1 and / or _CL domain.

In some embodiments, H1 is, if it contains at least one amino acid modification in the C _H1 domain, at least one of L1 and L2, C _L to a domain comprising at least one amino acid modification, and / or or H1 When at least one amino acid modification is included in the _VH domain, at least one of L1 and L2 includes at least one amino acid modification in the _VL domain.

  In some embodiments, H1, L1, H2 and / or L2 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications. In some embodiments, at least one of H1, H2, L1, and L2 is at least 2, 3, 4, 5, 6, 7, 8, 9 in at least one constant domain and / or at least one variable domain. Or 10 amino acid modifications.

  In some embodiments, when both L1 and L2 are co-expressed with at least one of H1 and H2, the relative pairing of at least one of the H1-L1 and H2-L2 heterodimer pairs is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 for each corresponding H1-L2 or H2-L1 heterodimer pair , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98 or more than 99%, the relative pairing of the modified H1-L1 or H2-L2 heterodimer pair is at least one amino acid Corresponding without qualification Greater than their relative pairing observed in 1-L1 or H2-L2 heterodimer pairs.

  In some embodiments, at least one of the first and second heterodimers has a thermal stability as measured by a melting temperature (Tm) as measured by DSF that does not have at least one amino acid modification. Within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ° C. of the Tm of the heterodimer. In some embodiments, the thermal stability of each heterodimer comprising at least one amino acid modification, as measured by the melting temperature (Tm) as measured by DSF, corresponds to having at least one amino acid modification. Within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ° C. of the Tm of the heterodimer. In some embodiments, the thermal stability of each heterodimer comprising at least one amino acid modification, as measured by the melting temperature (Tm) as measured by DSF, corresponds to having at least one amino acid modification. Within about 0, 1, 2, or 3 ° C. of the Tm of the heterodimer.

  In some embodiments, the affinity of each heterodimer for the binding antigen is determined by the affinity of the corresponding unmodified heterodimer for the same antigen as measured by surface plasmon resonance (SPR) or FACS. Within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times.

  In some embodiments, at least one of H1 and L1 comprises at least one amino acid modification that results in greater steric complementarity of amino acids when H1 pairs with L1 over L2. including. In some embodiments, at least one of H2 and L2 comprises at least one amino acid modification that results in greater steric complementarity of amino acids when H2 pairs with L2 rather than L1. including. In some embodiments, at least one of H1 and L1 comprises at least one amino acid modification that results in greater electrostatic complementarity between charged amino acids when H1 pairs with L1 over L2. Contains one domain. In some embodiments, at least one of H2 and L2 comprises at least one amino acid modification that results in greater electrostatic complementarity between charged amino acids when H2 pairs with L2 than L1. Contains one domain.

  In some embodiments, the at least one amino acid modification is a set of mutations as shown in at least one of the tables or examples.

In some embodiments, the construct further comprises an Fc comprising at least two C _H3 sequences, wherein the Fc comprises the first heterodimer and the second with or without one or more linkers. It binds to the heterodimer of

In some embodiments, the Fc is a human Fc, human IgG1 Fc, human IgA Fc, human IgG Fc, human IgD Fc, human IgE Fc, human IgM Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc. In some embodiments, the Fc is a heterodimeric Fc. In some embodiments, the Fc comprises one or more modifications in at least one of the _CH3 sequences. In some embodiments, the dimerized C _H3 sequence is about 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80 as measured by DSC. , 81, 82, 83, 84 or 85 ° C., or higher. In some embodiments, Fc, when produced, is about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92. , 93, 94, 95, 96, 97, 98, or a heterodimer formed with a purity greater than 99%, or Fc when expressed or via a single cell, about 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% It is a heterodimer formed with greater purity. In some embodiments, the Fc has one or more modifications in at least one of the _CH3 sequences that promotes the formation of a heterodimeric Fc with stability comparable to the wild type homodimeric Fc. Including. In some embodiments, the Fc further comprises at least one C _H2 sequence. In some embodiments, the Fc _CH2 sequence comprises one or more modifications. In some embodiments, the Fc comprises one or more modifications that facilitate selective binding of the Fc gamma receptor.

In some embodiments, Fc is
i) a heterodimeric IgG1 Fc having a modified L351Y_F405A_Y407V in the first Fc polypeptide and a modified T366L_K392M_T394W in the second Fc polypeptide;
ii) a heterodimeric IgG1 Fc having a modified L351Y_F405A_Y407V in the first Fc polypeptide and a modified T366L_K392L_T394W in the second Fc polypeptide;
iii) a heterodimeric IgG1 Fc having a modified T350V_L351Y_F405A_Y407V in the first Fc polypeptide and a modified T350V_T366L_K392L_T394W in the second Fc polypeptide;
iv) a heterodimeric IgG1 Fc having a modified T350V_L351Y_F405A_Y407V in the first Fc polypeptide and a modified T350V_T366L_K392M_T394W in the second Fc polypeptide; or v) having a modified T350V_L351Y_S400E_F40 The second Fc polypeptide comprises a heterodimeric IgG1 Fc having a modified T350V_T366L_N390R_K392M_T394W.

  In some embodiments, the Fc is bound to the heterodimer by one or more linkers, or the Fc is bound to H1 and H2 by one or more linkers. In some embodiments, the one or more linkers are one or more polypeptide linkers. In some embodiments, the one or more linkers comprise one or more antibody hinge regions. In some embodiments, the one or more linkers comprise one or more IgG1 hinge regions. In some embodiments, the one or more linkers include one or more modifications. In some embodiments, the one or more modifications to the one or more linkers facilitate selective binding of the Fc gamma receptor.

  In some embodiments, the at least one amino acid modification is at least one amino acid mutation, or the at least one amino acid modification is at least one amino acid substitution.

  In some embodiments, each sequence of H1, H2, L1, and L2 is derived from a human sequence.

  In some embodiments, the construct is multispecific or bispecific. In some embodiments, the construct is multivalent or divalent.

In some embodiments, the heterodimers described herein preferentially pair to form a bispecific antibody. For example, in some embodiments, the heavy chain polypeptide sequences H1 and H2 comprise a full length heavy chain sequence comprising a heavy chain constant domain (C _H1 domain), a C _H2 domain, and a C _H3 domain. In some embodiments, the percentage of heavy and light chains (eg, H1-L1: H2-L2) that pair exactly in a bispecific antibody is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, More than 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.

  Also described herein is an isolated polynucleotide or set of isolated polynucleotides comprising at least one sequence encoding a construct or heavy or light chain as described herein. In some embodiments, the polynucleotide or set of polynucleotides is cDNA.

  Also described herein are vectors or sets of vectors comprising one or more of the polynucleotides described herein or a set of polynucleotides. In some embodiments, the vector or set of vectors is selected from the group consisting of a plasmid, a multicistronic vector, a viral vector, a non-episomal mammalian vector, an expression vector, and a recombinant expression vector.

  Also described herein are isolated cells comprising a polynucleotide or set of polynucleotides described herein, or a vector or set of vectors described herein. In some embodiments, the cell is a hybridoma, Chinese hamster ovary (CHO) cell or HEK293 cell.

  Also described herein are pharmaceutical compositions comprising the constructs described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition is one or more selected from the group consisting of buffers, antioxidants, low molecular weight molecules, drugs, proteins, amino acids, carbohydrates, lipids, chelators, stabilizers and excipients. It further includes a plurality of substances.

  Also described herein is the use of a construct described herein or a pharmaceutical composition described herein in the treatment of a disease or disorder in a subject, or a cancer or vascular disease, or in the manufacture of a medicament. Is done.

  Also described herein is a method for treating a subject having a disease or disorder, or cancer or vascular disease, comprising administering to the subject a construct described herein or a composition described herein. The

  A method of obtaining a construct described herein from a host cell culture, comprising: (a) obtaining a host cell culture comprising at least one host cell comprising one or more nucleic acid sequences encoding the construct. And (b) recovering the construct from the host cell culture is also described herein.

  (A) obtaining H1, L1, H2, and L2, and (b) preferentially pairing H1 with L1 over L2, and preferentially pairing H2 with L2 over L1. Also described herein is a method of obtaining a construct as described herein, comprising (c) obtaining a construct.

  Obtaining a polynucleotide or set of polynucleotides encoding at least one construct and determining an optimal ratio of each of the polynucleotides or sets of polynucleotides for introduction into at least one host cell, comprising: The optimal ratio of H1, L1, H2 and L2 expression compared to the mismatched H1-L2 and H2-L1 heterodimer pairs formed during the expression of H1, L1, H2 and L2. Determining the amount of H1-L1 and H2-L2 heterodimer pairs formed and selecting a preferred optimal ratio, wherein a polynucleotide or polynucleotide has a preferred optimal ratio. Transfection of at least one host cell with a set of results in expression of the construct; and at least one host cell A method of preparing a construct as described herein comprising: transfection with an optimal ratio of polynucleotides or sets of polynucleotides; and culturing at least one host cell to express the construct. Are also described herein.

In some embodiments, selecting the optimal ratio is assessed by transfection in a transient transfection system. In some embodiments, transfection of at least one host cell with a preferred optimal ratio of polynucleotides or sets of polynucleotides results in optimal expression of the construct. In some embodiments, the construct comprises an Fc comprising at least two C _H3 sequences, wherein the Fc comprises the first heterodimer and the second with or without one or more linkers. Binds to the heterodimer. In some embodiments, the Fc is a heterodimer that optionally includes one or more amino acid modifications.

A first heterodimer and a second immunoglobulin heavy chain polypeptide sequence (H2) comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain polypeptide sequence (L1) ) And a second immunoglobulin light chain polypeptide sequence (L2), wherein the second heterodimer comprises data representing complementary mutations, each of H1 and H2 being at least a heavy chain variable domain (V _H Domain) and a heavy chain constant domain (C _H1 domain), and L1 and L2 each contain at least a light chain variable domain (V _L domain) and a light chain constant domain (C _L domain), and complementary mutation data A data set, wherein the set includes data representing the mutations or subsets of these mutations presented in the table or examples, and H1 A computer readable storage medium storing computer executable code that preferentially pairs with L1 over L2 and / or determines the likelihood that H2 preferentially pairs with L2 over L1 Described in the specification.

A first heterodimer and a second immunoglobulin heavy chain polypeptide sequence (H2) comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain polypeptide sequence (L1) ) And a second immunoglobulin light chain polypeptide sequence (L2), wherein the second heterodimer comprises data representing complementary mutations, each of H1 and H2 being at least a heavy chain variable domain (V _H Domain) and a heavy chain constant domain (C _H1 domain), wherein L1 and L2 each comprise at least a light chain variable domain ( _VL domain) and a light chain constant domain (C _L domain), complementary A data set containing data representing the mutations or subsets of these mutations presented in the tables or examples. And a computer processor determines the likelihood that H1 will preferentially pair with L1 over L2 and / or H2 preferentially pair with L2 over L1. A computer-implemented method for determining preferential pairing is also described herein. In some embodiments, the method further comprises producing a construct as described herein.

A method of producing a bispecific antigen binding polypeptide construct, wherein the bispecific construct comprises a first immunoglobulin heavy chain polypeptide sequence (H1) of a first monospecific antigen binding polypeptide. Immunoglobulin heavy chain polypeptide sequence (H2) of a first heterodimer and a second monospecific antigen binding polypeptide comprising a first immunoglobulin light chain polypeptide sequence (L1) And a second heterodimer comprising a second immunoglobulin light chain polypeptide sequence (L2), wherein H1 and H2 are at least a heavy chain variable domain (V _H domain) and a heavy chain constant domain (C includes _H1 domains), respectively L1 and L2, comprise at least a light chain variable domain _{(V L} domain) and a light chain constant domain _{(C L} domains), data described herein Introducing one or more complementary mutations of the tset into the first heterodimer and / or the second heterodimer; and the first heterodimer and the second heterodimer Are also co-expressed in at least one host cell to produce an expression product comprising the bispecific construct.

In some embodiments, the method further comprises determining the amount of bispecific construct in the expression product relative to other polypeptide products to select a preferred subset of complementary mutations. In some embodiments, the bispecific construct is greater than 70% compared to other polypeptide products (eg, 75%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or more than 99%). In some embodiments, the data set is a data set described herein. In some embodiments, the method adds an additional amino acid modification to at least one of H1, H2, L1, or L2 to increase the purity of the bispecific construct relative to other polypeptide products. Further comprising the step of: In some embodiments, the construct comprises an Fc comprising at least two C _H3 sequences, wherein the Fc comprises the first heterodimer and the second with or without one or more linkers. Binds to the heterodimer. In some embodiments, the Fc is a heterodimer that optionally includes one or more amino acid modifications. In some embodiments, the antigen binding polypeptide is an antibody, Fab or scFv.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, K145, D146, Q179 and S186, and L1 and / or L2 is Q124, S131. , V133, Q160, S176, T178 and T180 contain at least one amino acid modification or set of amino acid modifications. For example, in some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124R, L124E, K145M, K145T, D146N, Q179E, Q179K, S186R, and S186K. , L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q124E, S131R, S131K, V133G, Q160E, S176R, S176D, T178D, T178E and T180E. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of L124E, K145M, K145T and Q179E or a combination thereof, and L1 is a group consisting of S131R, S131K, V133G and S176R or a combination thereof. H2 includes an amino acid modification selected from the group consisting of L124R, D146N, Q179K, S186R and S186K or combinations thereof, and L2 includes Q124E, V133G, Q160E, S176D, T178D, Comprising an amino acid modification selected from the group consisting of T178E and T180E or combinations thereof. In some embodiments, H1 includes amino acid modifications L124E, K145T and Q179E, L1 includes amino acid modifications S131K, V133G and S176R, H2 includes amino acid modifications L124R and S186R, and L2 includes amino acid modifications V133G. , S176D and T178D.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, L143, K145, D146, Q179 and S186, and L1 and / or L2 is Q124. , V133, Q160, S176, T178, and T180 include at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124E, L124R, L143E, L143D, K145T, K145M, D146N, Q179K, S186R, and S186K. , L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q124K, Q124E, V133G, Q160K, S176R, S176D, T178E, T178K, T178R, T178D and T180E. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of L124E, L143E, L143D, K145T and K145M or combinations thereof, and L1 is Q124K, V133G, Q160K, S176R, T178K and T178R or H2 includes an amino acid modification selected from the group consisting of these combinations, H2 includes an amino acid modification selected from the group consisting of L124R, D146N, Q179K, S186R and S186K or a combination thereof, and L2 is Q124E, V133G, Comprising an amino acid modification selected from the group consisting of S176D, T178E, T178D and T180E or combinations thereof. In some embodiments, H1 includes amino acid modifications L124E, L143E, and K145T, L1 includes amino acid modifications Q124K, V133G, and S176R, H2 includes amino acid modifications L124R and Q179K, and L2 includes amino acid modifications V133G. , S176D and T178E. In some embodiments, H1 includes amino acid modifications L124E, L143E and K145T, L1 includes amino acid modifications Q124K, V133G and S176R, H2 includes amino acid modifications L124R and S186R, and L2 includes amino acid modifications V133G. , S176D and T178D.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications in Q39, L45, L124, L143, F122 and H172, and L1 and / or L2 is Q38 , P44, Q124, S131, V133, N137, S174, S176, and T178 include at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from Q39E, Q39R, L45P, F122C, L124E, L124R, L143F, H172T and H172R; And / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q38R, Q38E, P44F, Q124C, S131T, S131E, V133G, N137K, S174R, S176R, S176K, S176D, T178Y and T178D . In some embodiments, H1 comprises an amino acid modification selected from the group consisting of Q39E, L45P, F122C, L124E, L143F, H172T and H172R or combinations thereof, wherein L1 is Q38R, P44F, Q124C, S131T, Including an amino acid modification selected from the group consisting of V133G, N137K, S174R, S176R, S176K and T178Y or combinations thereof, and H2 includes an amino acid modification selected from the group consisting of Q39R, L124R and H172R or combinations thereof , L2 comprises an amino acid modification selected from the group consisting of Q38E, S131E, V133G, S176D and T178D or combinations thereof. In some embodiments, H1 comprises amino acid modifications Q39E and L124E, L1 comprises amino acid modifications Q38R, V133G and S176R, H2 comprises amino acid modifications Q39R and L124R, and L2 comprises amino acid modifications Q38E, V133G. And S176D. In some embodiments, H1 includes amino acid modifications L45P and L124E, L1 includes amino acid modifications P44F, V133G, and S176R, H2 includes amino acid modifications L124R, and L2 includes amino acid modifications V133G, S176D, and T178D. including. In some embodiments, H1 includes amino acid modifications L124E and L143F, L1 includes amino acid modifications V133G and S176R, H2 includes amino acid modifications L124R, and L2 includes amino acid modifications V133G, S176D, and T178D. . In some embodiments, H1 includes amino acid modifications F122C and L124E, L1 includes amino acid modifications Q124C, V133G, and S176R, H2 includes amino acid modifications L124R, and L2 includes amino acid modifications V133G and S176D. . In some embodiments, H1 comprises amino acid modifications L124E and H172T, L1 comprises amino acid modifications V133G, N137K, S174R and S176R, H2 comprises amino acid modifications L124R and H172R, and L2 comprises amino acid modifications V133G. , S176D and T178D.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, A125, H172 and K228, and L1 and / or L2 is S121, V133, N137. , S174, S176 and T178 include at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124E, L124R, A125S, A125R, H172R, H172T and K228D, and (ii) L1 and L2 comprises at least one amino acid modification or set of amino acid modifications selected from S121K, V133G, N137K, S174R, S176K, S176R, S176D and T178D. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of L124E, A125S, H172R and K228D or combinations thereof, and L1 is selected from the group consisting of S121K, V133G and S176R or combinations thereof Wherein H2 comprises an amino acid modification selected from the group consisting of L124R, A125R and H172T or combinations thereof, and L2 is a group consisting of V133G, N137K, S174R, S176D and T178D or combinations thereof An amino acid modification selected from In some embodiments, H1 includes amino acid modifications L124E and K228D, L1 includes amino acid modifications S121K, V133G, and S176R, H2 includes amino acid modifications L124R and A125R, and L2 includes amino acid modifications V133G and S176D. including. In some embodiments, H1 includes amino acid modifications L124E and H172R, L1 includes amino acid modifications V133G and S176R, H2 includes amino acid modifications L124R and H172T, and L2 includes amino acid modifications V133G, S174R, and S176D. including.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, A139 and V190, and L1 and / or L2 are at F116, V133, L135 and S176. It includes at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124E, L124R, A139W, A139G and V190A, and L1 and / or L2 is F116A, It comprises at least one amino acid modification or set of amino acid modifications selected from V133G, L135V, L135W, S176R and S176D. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of L124E and A139W or a combination thereof, and L1 is selected from the group consisting of F116A, V133G, L135V and S176R or a combination thereof. An amino acid modification, wherein H2 comprises an amino acid modification selected from the group consisting of L124R, A139G and V190A or a combination thereof, and L2 is an amino acid modification selected from the group consisting of V133G, L135W and S176D or a combination thereof including. In some embodiments, H1 comprises amino acid modifications L124E and A139W, L1 comprises amino acid modifications F116A, V133G, L135V and S176R, H2 comprises amino acid modifications L124R, A139G and V190A, and L2 comprises amino acids Modifications V133G, L135W and S176D are included.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at Q39, L45, K145, H172, Q179 and S186, and L1 and / or L2 is Q38 , P44, Q124, S131, Q160, T180, and C214 include at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from Q39E, Q39R, L45P, K145T, H172R, Q179E, and S186R, and L1 and / or L2 Comprises at least one amino acid modification or set of amino acid modifications selected from Q38R, Q38E, P44F, Q124E, S131K, Q160E, T180E and C214S. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of Q39E, L45P, K145T, H172R and Q179E or combinations thereof, and L1 is a group consisting of Q38R, P44F and S131K or combinations thereof H2 includes an amino acid modification selected from the group consisting of Q39R, H172R and S186R or combinations thereof, and L2 includes Q38E, Q124E, Q160E, T180E and C214S or combinations thereof An amino acid modification selected from the group consisting of: In some embodiments, H1 comprises amino acid modifications Q39E, K145T and Q179E, L1 comprises amino acid modifications Q38R and S131K, H2 comprises amino acid modifications Q39R and S186R, and L2 comprises amino acid modifications Q38E, Q124E. Q160E and T180E. In some embodiments, H1 comprises amino acid modifications L45P, K145T, H172R and Q179E, L1 comprises amino acid modifications P44F and S131K, H2 comprises amino acid modifications H172R and S186R, and L2 comprises amino acid modification Q124E. Q160E and T180E.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at A139, L143, K145, Q179 and V190, and L1 and / or L2 is F116, Q124. , L135, Q160, T178 and T180 contain at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from A139W, A139G, L143E, K145T, Q179E, Q179K and V190A, and L1 and / or L2 Comprises at least one amino acid modification or set of amino acid modifications selected from F116A, Q124R, Q124E, L135V, L135W, Q160E, T178R and T180E. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of A139W, L143E, K145T and Q179E or combinations thereof, and L1 is a group consisting of F116A, Q124R, L135V and T178R or combinations thereof H2 comprises an amino acid modification selected from the group consisting of A139G, Q179K and V190A or combinations thereof, and L2 is a group consisting of Q124E, L135W, Q160E and T180E or combinations thereof An amino acid modification selected from In some embodiments, H1 comprises amino acid modifications A139W, L143E, K145T and Q179E, L1 comprises amino acid modifications F116A, Q124R, L135V and T178R, H2 comprises amino acid modification Q179K, and L2 comprises amino acid modifications Modification Q124E, L135W, Q160E and T180E are included.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at Q39, L143, K145, D146, H172 and Q179, and L1 and / or L2 is Q38. , Q124, Q160, T178 and T180 include at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from Q39E, Q39R, L143E, K145T, D146G, H172R, Q179E and Q179K, and L1 and / or Or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q38R, Q38E, Q124R, Q124E, Q160K, Q160E, T178R and T180E. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of Q39E, L143E, K145T, H172R and Q179E or combinations thereof, and L1 is from Q38R, Q124R, Q160K and T178R or combinations thereof H2 includes an amino acid modification selected from the group consisting of Q39R, H172R and Q179K or combinations thereof, and L2 includes Q38E, Q124E, D146G, Q160E and T180E or these Including amino acid modifications selected from the group consisting of combinations. In some embodiments, H1 comprises amino acid modifications Q39E, L143E, K145T and Q179E, L1 comprises amino acid modifications Q38R, Q124R, Q160K and T178R, H2 comprises amino acid modifications Q39R, H172R and Q179K, L2 contains amino acid modifications Q38E, Q124E, Q160E and T180E.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L45, L143, K145, D146, H172 and Q179, and L1 and / or L2 is Q38. , P44, Q124, N137, Q160, S174, T178, T180, and C214 include at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L45P, L143E, K145T, D146G, H172R, H172T, Q179E and Q179K, and (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q38E, P44F, Q124R, Q124E, N137K, Q160K, Q160E, S174R, T178R, T180E and C214S. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of L45P, L143E, K145T, H172R and Q179E or combinations thereof, and L1 is from P44F, Q124R, Q160K and T178R or combinations thereof H2 includes an amino acid modification selected from the group consisting of D146G, H172R, H172T and Q179K or combinations thereof, and L2 includes Q38E, Q124E, N137K, Q160E, S174R, Comprising an amino acid modification selected from the group consisting of T180E and C214S or combinations thereof. In some embodiments, H1 comprises amino acid modifications L45P, L143E and K145T, L1 comprises amino acid modifications P44F, Q124R, Q160K and T178R, H2 comprises amino acid modifications D146G and Q179K, and L2 comprises amino acid modifications Modification Q38E, Q124E, Q160E and T180E are included. In some embodiments, H1 comprises amino acid modifications L143E, K145T and H172R, L1 comprises amino acid modifications Q124R, Q160K and T178R, H2 comprises amino acid modifications H172T and Q179K, and L2 comprises amino acid modification Q124E. , Q160E, N137K, S174R and T180E.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, L143, K145 and Q179, wherein L1 and / or L2 is Q124, S131, V133. , S176, T178 and T180 contain at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124W, L124A, L143E, L143F, K145T, Q179E, and Q179K, and L1 and / or L2 Comprises at least one amino acid modification or set of amino acid modifications selected from Q124R, Q124K, Q124E, S131K, V133A, V133W, S176T, T178R, T178L, T178E and T180E. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of L124W, L143E, K145T and Q179E or combinations thereof, and L1 is Q124R, Q124K, S131K, V133A, S176T, T178R and T178L or Comprising an amino acid modification selected from the group consisting of these combinations, H2 comprising an amino acid modification selected from the group consisting of L124A, L143F and Q179K or a combination thereof, L2 is Q124E, V133W, S176T, T178L, Comprising an amino acid modification selected from the group consisting of T178E and T180E or combinations thereof. In some embodiments, H1 includes amino acid modifications L124W, L143E, K145T, and Q179E, L1 includes amino acid modifications Q124R, V133A, S176T, and T178R, and H2 includes amino acid modifications L124A, L143F, and Q179K; L2 includes amino acid modifications Q124E, V133W, S176T, T178L and T180E.

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at A139, L143, K145, Q179 and S186, and L1 and / or L2 is F116, Q124. , V133, Q160, T178 and T180 contain at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 is at least one amino acid modification or amino acid modification selected from A139C, L143E, L143D, L143R, L143K, K145T, Q179E, Q179D, Q179R, Q179K, S186K and S186R And L1 and / or L2 is at least one amino acid modification or set of amino acid modifications selected from F116C, Q124R, Q124K, Q124E, V133E, V133D, Q160K, Q160E, T178R, T178K, T178E and T180E including. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of A139C, L143E, L143D, K145T, Q179E and Q179D or combinations thereof, and L1 is F116C, Q124R, Q124K, Q160K, T178R and H2 includes an amino acid modification selected from the group consisting of T178K or a combination thereof, and H2 includes an amino acid modification selected from the group consisting of L143R, L143K, Q179R, Q179K, S186K and S186R or a combination thereof, and L2 is Comprising an amino acid modification selected from the group consisting of Q124E, V133E, V133D, Q160E, T178E and T180E or combinations thereof. In some embodiments, H1 includes amino acid modifications A139C, L143E, K145T, and Q179E, L1 includes amino acid modifications F116C, Q124R, and T178R, H2 includes amino acid modification Q179K, and L2 includes amino acid modification Q124E. Q160E and T180E. In some embodiments, H1 includes amino acid modifications L143E, K145T, and Q179E, L1 includes amino acid modifications Q124R and T178R, H2 includes amino acid modification S186K, and L2 includes amino acid modifications Q124E, Q160E, and T178E. including. In some embodiments, H1 includes amino acid modifications L143E, K145T, and Q179E, L1 includes amino acid modifications Q124R and T178R, H2 includes amino acid modifications L143R, and L2 includes amino acid modifications Q124E and V133E. .

  In some embodiments of the construct, H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, L143, K145, D146, Q179, S186 and S188, wherein L1 and / or L2 is , Q124, S131, V133, Q160, S176, T178, and T180 include at least one amino acid modification or set of amino acid modifications. In some embodiments, H1 and / or H2 is at least one amino acid modification selected from L124A, L143A, L143R, L143E, L143K, K145T, D146G, Q179R, Q179E, Q179K, S186R, S186K, and S188L Including a set of amino acid modifications, L1 and / or L2 is selected from Q124R, Q124E, S131E, S131T, V133Y, V133W, V133E, V133D, Q160E, Q160K, Q160M, S176L, T178R, T178E, T178F, T178Y and T180E At least one amino acid modification or set of amino acid modifications. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of L143E, K145T, Q179E and S188L or combinations thereof, and L1 is selected from the group consisting of Q124R, Q160K and T178R or combinations thereof H2 includes an amino acid modification selected from the group consisting of L124A, L143A, L143R, L143K, D146G, Q179R, Q179K, S186R and S186K, or a combination thereof, and L2 includes Q124E, S131E, Selected from the group consisting of S131T, V133Y, V133W, V133E, V133D, Q160E, Q160M, S176L, T178E, T178F, T178Y and T180E or combinations thereof It is, including the amino acid modification. In some embodiments, H1 comprises amino acid modifications L143E, K145T, Q179E and S188L, L1 comprises amino acid modifications Q124R and T178R, H2 comprises amino acid modification S186K, and L2 comprises amino acid modifications Q124E, S176L. And T180E. In some embodiments, H1 comprises amino acid modifications L143E, K145T, Q179E and S188L, L1 comprises amino acid modifications Q124R and T178R, H2 comprises amino acid modification S186K, and L2 comprises amino acid modifications Q124E, S131T. , T178Y and T180E. In some embodiments, H1 includes amino acid modifications L143E and K145T, L1 includes amino acid modifications Q124R, Q160K, and T178R, H2 includes amino acid modification S186K, and L2 includes amino acid modification S131E. In some embodiments, H1 comprises amino acid modifications L143E and K145T, L1 comprises amino acid modification Q124R, H2 comprises amino acid modification L143R, and L2 comprises amino acid modifications Q124E and V133E.

  In some embodiments of the construct, H1 comprises at least one amino acid modification or set of amino acid modifications at F122 and C233, and L1 comprises at least one amino acid modification or set of amino acid modifications at Q124 and C214. . In some embodiments, H1 comprises at least one amino acid modification or set of amino acid modifications selected from F122C and C233S, and L1 is at least one amino acid modification or amino acid modification selected from Q124C and C214S. Includes a set of In some embodiments, H1 comprises an amino acid modification selected from the group consisting of F122C and C233S or a combination thereof, and L1 comprises an amino acid modification selected from the group consisting of Q124C and C214S or a combination thereof. , H2 contains a wild type or unmodified amino acid sequence and L2 contains a wild type or unmodified amino acid sequence. In some embodiments, H1 comprises amino acid modifications F122C and C233S, L1 comprises amino acid modifications Q124C and C214S, H2 comprises a wild type or unmodified amino acid sequence, and L2 is wild type or unmodified. Contains amino acid sequence.

  In some embodiments, the construct is a SMCA design 9561-9095_1, 9561-9095_2, 9121-9373_1, 9121-9373_2, 9116-9349_1, 9116-9349_2, 9134-9521_1, 9134-9521_2, in the tables herein. 9286-9402_1, 9286-9402_2, 9667-9830_1, 9667-9830_2, 9696-9848_1, 9696-9848_2, 9060-9756_1, 9060-9756_2, 9682-9740_1, 9682-9740_2, 9049-9759_2, 9849-9759_2 Contains amino acid modifications selected from 9823_1 and 9820-9823_2. In some embodiments, the construct is an amino acid selected from SMCA designs 9327-6054_1, 9815-9825_1, 9815-9825_2, 9587-9735_1, 9587-9735_2, 3522_1, 3522_2, 3519_1 and 3519_2 in the tables herein. Includes modifications.

  In some embodiments, H1 and / or H2 does not contain an amino acid modification at position Q179. In some embodiments, H1 does not contain amino acid modification Q179E and / or H2 does not contain amino acid modification Q179K. In some embodiments, L1 does not contain an amino acid modification at position S131. In one embodiment, L1 does not contain the amino acid modification S131K. In some embodiments, L2 does not contain an amino acid modification at position T180. In one embodiment, L2 does not include the amino acid modification T180E. In some embodiments, the construct does not include a combination of amino acid modifications where H1 includes Q179E, L1 includes S131K, H2 includes Q179K, and L2 includes T180E.

  In some embodiments, H1 does not contain an amino acid modification at positions Q39 and / or Q179. In some embodiments, H1 does not include amino acid modifications Q39E and / or Q179E. In some embodiments, L1 does not contain an amino acid modification at position Q160. In one embodiment, L1 does not contain the amino acid modification Q160K. In some embodiments, H2 does not contain an amino acid modification at position Q179. In one embodiment, H2 does not include the amino acid modification Q179K. In some embodiments, L2 does not contain an amino acid modification at positions Q38, Q160 and / or T180. In one embodiment, L2 does not include amino acid modifications Q38E, Q160E, and / or T180E. In some embodiments, the construct comprises a combination of amino acid modifications, wherein H1 includes Q39E and / or Q179E, L1 includes Q160K, H2 includes Q179K, and L2 includes Q38E, Q160E, and / or T180E. Absent. For example, in some embodiments, the construct comprises (i) H1 comprises Q179E, L1 comprises Q160K, H2 comprises Q179K, L2 comprises Q160E and T180E, (ii) H1 comprises Q39E and Q179E. L1 includes Q160K, H2 includes Q179K, L2 includes Q38E, Q160E and T180E, or (iii) H1 includes Q39E, L1 includes Q160K, H2 includes Q179K, L2 includes Q38E, Does not include a combination of amino acid modifications, including Q160E and T180E.

  In some embodiments, H1 does not contain an amino acid modification at position Q179. In some embodiments, H1 does not include the amino acid modification Q179K or Q179E. In some embodiments, L1 does not contain an amino acid modification at position Q160 and / or T180. In one embodiment, L1 does not include amino acid modifications Q160E, Q160K, and / or T180E. In some embodiments, H2 does not contain an amino acid modification at position Q179. In one embodiment, H2 does not include the amino acid modification Q179K or Q179E. In some embodiments, L2 does not contain an amino acid modification at position Q160 and / or T180. In one embodiment, L2 does not include amino acid modifications Q160K, Q160E, and / or T180E. In some embodiments, the construct comprises H1 comprises Q179K or Q179E, L1 comprises Q160E, Q160K and / or T180E, H2 comprises Q179K or Q179E, and L2 comprises Q160K, Q160E and / or T180E. Does not include combinations of amino acid modifications.

  In some embodiments, H1 and / or H2 does not contain an amino acid modification at position Q179. In some embodiments, H1 does not contain amino acid modification Q179K and / or H2 does not contain amino acid modification Q179E. In some embodiments, L1 does not contain an amino acid modification at position T180. In one embodiment, L1 does not include the amino acid modification T180E. In some embodiments, L2 does not contain an amino acid modification at position S131. In one embodiment, L2 does not contain the amino acid modification S131K. In some embodiments, the construct does not include a combination of amino acid modifications where H1 includes Q179K, L1 includes T180E, H2 includes Q179E, and L2 includes S131K.

  In some embodiments, H1 does not contain an amino acid modification at position Q179. In some embodiments, H1 does not include amino acid modification Q179E. In some embodiments, L1 does not contain an amino acid modification at position Q160. In one embodiment, L1 does not contain the amino acid modification Q160K. In some embodiments, H2 does not contain an amino acid modification at position Q179. In one embodiment, H2 does not include the amino acid modification Q179K. In some embodiments, L2 does not contain an amino acid modification at position T180. In one embodiment, L2 does not include the amino acid modification T180E. In some embodiments, the construct does not include a combination of amino acid modifications where H1 includes Q179E, L1 includes Q160K, H2 includes Q179K, and L2 includes T180E.

  In some embodiments, H1 does not contain an amino acid modification at position A139. In some embodiments, H1 does not include amino acid modification A139C. In some embodiments, L1 does not contain an amino acid modification at position F116. In one embodiment, L1 does not include the amino acid modification F116C. In some embodiments, the construct does not include a combination of amino acid modifications where H1 includes A139C and L1 includes F116C.

  In some embodiments, the construct does not include a native disulfide linkage between the heavy and light chains. For example, in some embodiments, the cysteine at position 214 of L1 and / or L2 is modified to another amino acid. In some embodiments, L1 and / or L2 comprises the amino acid modification C214S. In some embodiments, the cysteine at position 233 of H1 and / or H2 is modified to another amino acid. In one embodiment, H1 and / or H2 comprises the amino acid modification C233S.

  The embodiments described herein are applicable to Fab format and full antibody format constructs.

The D3H44 heavy and light chain amino acid sequences aligned to the basic human germline sequence are shown for variable, constant and J region segments (shown in the figure: ^* sequence identity). FIG. 1A shows the human VH germline subgroup (one representative sequence is displayed in each family). Sequence identity based on alignment of D3H44 to VH3 and IGHJ3 ^* 02. Human kappa VL germline subgroups are shown (one representative sequence is displayed in each family). Sequence identity based on the alignment of D3H44 to VKI and IGKJ1 ^* 01. Human lambda VL germline subgroups are shown (one representative sequence is displayed in each family). Sequence identity based on the alignment of D3H44 to VL1 and IGLJ1 ^* 01. The human CH1 allele sequence is shown. The human kappa and lambda allele sequences are shown. FIG. 5 shows a flowchart for computational modeling to identify key interface residues and to design preferential heavy chain-light chain pairs. FIG. 3 shows an exemplary set of H1, L1, H2, and L2 chains designed such that H1 preferentially pairs with L1 over L2, and H2 preferentially pairs with L2 over L1. A diagram representing the three-dimensional crystal structure of the variable region heavy and light chain interface is presented. Mutations introduced at the interface achieve electrostatic and steric complementarity to preferentially form the absolute pairs H1-L1 and H2-L2, respectively. On the other hand, unfavorable steric and electrostatic incompatibility exists in inaccurate pairs, leading to reduced pairing tendency and reduced stability for incompatible pairs. A high-level schematic for genetic engineering manipulation requirements to form bispecific Mabs (monoclonal antibodies) and the assay requirements necessary to quantify heavy chain light chain pairs are described. The design goal of genetically engineering a high purity bispecific Mab (ie, with little or no association of mismatched HL) is two unique heavy chains and a unique cognate light chain. Preferential pairing with the chain can be achieved by rational genetic engineering (via the introduction of specific amino acid mutations). This process is shown schematically, where H1 is genetically engineered to preferentially pair with L1, not L2. Similarly, H2 is genetically engineered to preferentially pair with L2, not L1. Biological Mab design for experimental selection requires an assay that can simultaneously quantify H1-L1: H1-L2 and H2-L2: H2-L1. These assay requirements can be simplified by assuming that each bispecific Fab arm can be engineered independently. In this case, the assay need only quantify H1-L1: H1-L2 or H2-L2: H2-L1, and not both simultaneously. Provides a schematic depiction of how heavy and light chains are tagged and how preferential pairing is determined. In this schematic, the circle represents a cell transfected with three constructs. The expression product is secreted from the cells and the supernatant (SPNT) flows to a detection device, in this case an SPR chip. Estimating a quantitative estimate of preferential pairing of heavy chains to two light chains based on the detection level of two different tags fused to the two light chains competing for heavy chain pairing Can do. In each cluster, a box plot of mean LCCA performance values with Fab heterodimer pairing: mispairing is at least 86:14 is shown. 2 shows representative UPLC-SEC profiles of A) WT Fab heterodimer and B) representative designed Fab heterodimers (H1L1 Fab components of LCCA designs 9735, 9737 and 9740). 2 shows possible heavy chain association products that can be predicted when two different light chains are co-expressed in a cell with two different heavy chains. Preferential pairing is assessed by use of SMCA (monoclonal antibody competition assay). Figure 2 shows the bias / strand utilization preference in the D3H44 / trastuzumab bispecific system. Chain utilization was evaluated in different species observed by LC-MS. The X-axis represents the H1: H2: L1: L2 DNA ratio and the Y-axis shows the corresponding rate of each strand in different transfection experiments. In the equilibrium system, all H chains and L chains represent 25%. A bias towards the utilization of one light chain is observed across all bispecific systems. Figure 2 shows the bias / strand utilization preference in the D3H44 / cetuximab bispecific system. Chain utilization was evaluated in different species observed by LC-MS. The X-axis represents the H1: H2: L1: L2 DNA ratio and the Y-axis shows the corresponding rate of each strand in different transfection experiments. In the equilibrium system, all H chains and L chains represent 25%. A bias towards the utilization of one light chain is observed across all bispecific systems. Figure 2 shows the bias / strand utilization preference in a trastuzumab / cetuximab bispecific system. Chain utilization was evaluated in different species observed by LC-MS. The X-axis represents the H1: H2: L1: L2 DNA ratio and the Y-axis shows the corresponding rate of each strand in different transfection experiments. In an equilibrium system, all H and L chains represent 25%. A bias towards the utilization of one light chain is observed across all bispecific systems. FIG. 10 shows representative UPLC-SEC profiles for WT and modified heterodimeric antibodies, respectively. FIG. 10a refers to D3H44 / Trastuzumab WT. 9060-9756_1. Refers to D3H44 / cetuximab WT. 9820-9823_1. Refers to trastuzumab / cetuximab WT. 9696-9848_1. Percent change of correctly matched Fab components (%) for all mismatched Fab components utilizing the same heavy chain (% of D3H44 / trastuzumab and D3H44 / cetuximab) in the modified bispecific antibody sample per cluster Boxes of H1: L1 change for all H1 species for wild type, H2: L2 change for all H2 species for wild type trastuzumab / cetuximab), and the desired bispecific antibody rate change for wild type A diagram is shown. The percent change of precisely matched Fab components for all mispaired Fab components utilizing the same heavy chain per cluster is shown in the D3H44 / trastuzumab system. The change in the rate of desired bispecific antibody to wild type from cluster to cluster is shown in the D3H44 / trastuzumab system. Percent change of correctly matched Fab components (%) for all mismatched Fab components utilizing the same heavy chain (% of D3H44 / trastuzumab and D3H44 / cetuximab) in the modified bispecific antibody sample per cluster Boxes of H1: L1 change for all H1 species for wild type, H2: L2 change for all H2 species for wild type trastuzumab / cetuximab), and the desired bispecific antibody rate change for wild type A diagram is shown. The percent change in precisely matched Fab components for all mispaired Fab components utilizing the same heavy chain per cluster is shown in the D3H44 / cetuximab system. The change in the ratio of desired bispecific antibody to wild type from cluster to cluster is shown in the D3H44 / cetuximab system. Percent change of correctly matched Fab components (%) for all mismatched Fab components utilizing the same heavy chain (% of D3H44 / trastuzumab and D3H44 / cetuximab) in the modified bispecific antibody sample per cluster Boxes of H1: L1 change for all H1 species for wild type, H2: L2 change for all H2 species for wild type trastuzumab / cetuximab), and the desired bispecific antibody rate change for wild type A diagram is shown. The percent change in precisely matched Fab components for all mispaired Fab components utilizing the same heavy chain per cluster is shown in the trastuzumab / cetuximab system. The change in the ratio of desired bispecific antibody to wild type from cluster to cluster is shown in the trastuzumab / cetuximab system. Shows the percent change of precisely matched Fab components for all mis-matched Fab components utilizing the same heavy chain per cluster across all bispecific systems. Shown is the change in the rate of desired bispecific antibodies relative to the wild type for each cluster. Note that the values reported in FIG. 11 also include putative changes in modified bispecific antibody samples for which the corresponding wild type construct was not evaluated by SMCA. 2 illustrates a method for preparing bispecific antibodies using a library of absolute mutation pairs provided herein.

  An antigen-binding polypeptide construct (heterodimer) that can comprise a first heterodimer and a second heterodimer, each heterodimer comprising an immunoglobulin heavy chain or fragment thereof and an immunoglobulin light chain. (Also referred to as a mer pair) is provided herein. Both heterodimers have one or more amino acid modifications in immunoglobulin heavy chain constant domain 1 (CH1) and one or more amino acid modifications in immunoglobulin light chain constant domain (CL), immunoglobulin heavy chain variable One or more amino acid modifications in the domain (VH) and one or more amino acid modifications in the immunoglobulin light chain variable domain (VL), or the amino acid modifications described above in both the heavy and light chain constant and variable domains. Combinations can be included. The modified amino acid is typically part of the light and heavy chain interface, with the heavy chain of the first heterodimer preferentially paired with one light chain but not the other. Modified to not match, creating a preferential pairing between each heavy chain and the desired light chain. Similarly, the heavy chain of the second heterodimer can preferentially pair with the second light chain rather than the first.

  As indicated above, certain combinations of amino acid modifications described herein facilitate preferential pairing of heavy chains with certain light chains, thereby negligible Bispecific monoclonals so that there is minimal need to purify the desired heterodimer from unwanted or mismatched products Antibody (Mab) expression can occur. Heterodimers can exhibit thermal stability comparable to heterodimers without amino acid modifications and demonstrate binding affinity for antigens comparable to heterodimers without amino acid modifications You can also.

  Use the first and second heterodimer designs to target two different therapeutic targets, or target two distinct epitopes (overlapping or non-overlapping) within the same antigen Specific antibodies can be made.

  Methods for preparing heterodimer pairs are also provided herein.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In the event that there are a plurality of definitions for terms herein, those in this section prevail. If a reference is made to a URL or other such identifier or address, such identifiers may change and certain information on the Internet may appear or disappear, but equivalent information is Can be found by searching the internet. References to these are evidence that such information is available and popular.

  It should be understood that the foregoing general description and the following detailed description are for purposes of illustration and description only and are not intended to limit any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise.

  In this description, any concentration range, percentage range, ratio range, or integer range, unless indicated otherwise, is any integer within the recited range, if appropriate, a fraction thereof (eg, an integer of 10 minutes) It should be understood to include values of 1 and 1/100). As used herein, “about” means ± 10% of the indicated range, value, sequence or structure, unless otherwise indicated. The terms “a” and “an” as used herein are “one” of the listed components unless otherwise indicated or determined from context. It should be understood to refer to “or plural”. It should be understood that use of an option (eg, “or”) means any one, both, or any combination of options. As used herein, the terms “include” and “comprise” are used interchangeably. In addition, individual single chain polypeptides or immunoglobulin constructs derived from various combinations of structures and substituents described herein are described separately for each single chain polypeptide or heterodimer. It should be understood that it is disclosed in this application to the same extent as it is. Accordingly, the selection of specific components to form individual single chain polypeptides or heterodimers is within the scope of this disclosure.

  The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents or documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and treaty documents, are hereby incorporated by reference in their entirety for any purpose. Specifically incorporated into the description.

  It should be understood that the methods and compositions described herein are not limited to the particular methodologies, protocols, cell lines, constructs and reagents described herein, and that they can vary. is there. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein. It should also be understood that it is limited only by the scope of the appended claims.

  All publications and patents mentioned in this specification include constructs and methodologies described in the publication that can be used in connection with, for example, the methods, compositions, and compounds described herein. For purposes of description and disclosure, the entirety of which is incorporated herein by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. It should be understood herein that the invention described herein is not to be construed as an admission that it is not entitled to antedate such disclosure by virtue of prior invention or for any other reason. Nor.

  In this application, amino acid names and atom names (eg, N, O, C, etc.) are used as defined in Protein DataBank (PDB) (www.pdb.org), which is the IUPAC nomenclature. Eur. J. Biochem., 138, 9 with amendments according to the method (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom names, etc.), Eur. J. Biochem., 152, 1 (1985). -37 (1984) The term “amino acid residue” refers to the 20 naturally occurring amino acids: alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D). , Glutamic acid (Glu ma E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V) ), Tryptophan (Trp or W) and tyrosine (Tyr or Y) residues are mainly intended to indicate the amino acid residues contained in the group.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description directed to a peptide and a description directed to a protein, and vice versa. This term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full length proteins in which amino acid residues are linked by covalent peptide bonds.

  The term “nucleotide sequence” or “nucleic acid sequence” is intended to indicate that two or more nucleotide molecules extend continuously. The nucleotide sequence can be genomic, cDNA, RNA, semi-synthetic or synthetically derived, or any combination thereof.

  “Cells”, “host cells”, “cell lines” and “cell cultures” are used interchangeably herein, and such terms all refer to the passages brought about by cell growth or culture. It should be understood to include. “Transformation” and “transfection” are used interchangeably to refer to the process of introducing a nucleic acid sequence into a cell.

  The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are 20 amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, Tyrosine and valine), and pyrrolysine and selenocysteine. Amino acid analogs have the same basic chemical structure as a naturally occurring amino acid, namely carbon, carboxyl, amino and R groups bonded to hydrogen, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, etc. Refers to a compound. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. References to amino acids include, for example, naturally occurring proteinogenic L amino acids, D-amino acids, chemically modified amino acids such as amino acid variants and derivatives, naturally occurring non-proteinogenic amino acids such as alanine, ornithine, and the like, and Chemically synthesized compounds having properties known in the art to be characteristic of amino acids are included. Examples of non-naturally occurring amino acids include □ -methyl amino acids (eg, methylalanine), D-amino acids, histidine-like amino acids (eg, 2-amino-histidine, hydroxy-histidine, homohistidine), additional methylene side Examples include, but are not limited to, amino acids in the chain ("homo" amino acids), and amino acids in which the side chain carboxylic acid functionality is replaced by a sulfonic acid group (eg, cysteic acid). Incorporation of synthetic native amino acids, substituted amino acids or unnatural amino acids, including one or more D-amino acids, into the proteins of the invention can be beneficial in a number of different ways. D-amino acid-containing peptides and the like exhibit increased stability in vitro or in vivo compared to their L-amino acid-containing counterparts. Thus, the construction of peptides and the like that incorporate D-amino acids may be particularly useful when greater intracellular stability is desired or required. More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, so that when such properties are desired, the improved bioavailability of the molecule and extended lifetime in vivo. Bring. In addition, D-peptides and the like are not efficiently processed by the restricted presentation of major histocompatibility complex class II to T helper cells, thus eliciting a humoral immune response throughout the organism Less likely.

  Amino acids are referred to herein by the commonly known three letter codes or by the one letter codes recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referenced by generally accepted single letter codes.

  “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a “conservatively modified variant” refers to a nucleic acid that encodes the same or substantially the same amino acid sequence, or a substantially identical sequence if the nucleic acid does not encode an amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art can modify each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) to yield a functionally identical molecule. Recognize Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

  For amino acid sequences, one of ordinary skill in the art will recognize individual substitutions, deletions or deletions to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence. It is recognized that addition is a “conservatively modified variant” in which the change results in deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those skilled in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphisms, variants, interspecies homologs and alleles of the present invention.

Conservative substitution tables providing functionally similar amino acids are known to those skilled in the art. The following eight groups contain amino acids with conservative substitutions for each other:
Alanine (A), glycine (G);
Aspartic acid (D), glutamic acid (E);
Asparagine (N), glutamine (Q);
Arginine (R), lysine (K);
Isoleucine (I), leucine (L), methionine (M), valine (V);
Phenylalanine (F), tyrosine (Y), tryptophan (W);
Serine (S), threonine (T); and cysteine (C), methionine (M).
(See, for example, Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co., Second Edition (December 1993)).

  The term “identical” or “identity” rate refers to two or more sequences or subsequences that are the same in the context of two or more nucleic acid or polypeptide sequences. Sequences were measured when compared and aligned to the maximum extent over the comparison range, or using one of the following sequence comparison algorithms (or other algorithms available to those skilled in the art) over the specified region. Or by manual alignment and visual inspection, the percentage of amino acid residues or nucleotides that are the same (ie, at least about 50%, 55%, 60%, 65%, 70%, 75%, over the specified region) 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity). is there. This definition is also referred to as the complement of the test sequence. Identity exists over a region of at least about 50 amino acids or nucleotides in length, or over a region of 75-100 amino acids or nucleotides in length, or over the entire sequence of a polynucleotide or polypeptide if not specified. Can do. A polynucleotide encoding a polypeptide of the present invention, including homologues from species other than human, can be obtained by subjecting the library to stringent hybridization conditions using a labeled probe having the polynucleotide sequence of the present invention or a fragment thereof. Can be obtained by a process comprising screening and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art.

  A derivative or variant of a polypeptide shares “homology” or “homology” with a peptide if the amino acid sequence of the derivative or variant has at least 50% identity with the sequence of 100 amino acids of the original peptide. Is said. In certain embodiments, the derivative or variant is at least 75% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 85% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of the derivative is at least 90% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 95% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 99% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative.

  As used herein, an “isolated” polypeptide or construct refers to a construct or polypeptide that has been identified and separated and / or recovered from a component of the natural cell culture environment. Contaminating components of the natural environment are materials that typically interfere with the diagnostic or therapeutic use of heteromultimers and can include enzymes, hormones and other proteinaceous or non-proteinaceous solutes.

  In certain embodiments, as used herein, an “isolated” antigen-binding polypeptide construct described herein is identified and separated from a component of a natural cell culture environment; And / or heterodimer pairs or “isolated” heterodimer pairs, including recovered heterodimers or heterodimer pairs. Contaminating components of the natural environment are materials that interfere with the diagnostic or therapeutic use of heterodimers or antigen-binding polypeptide constructs and can include enzymes, hormones and other proteinaceous or non-proteinaceous solutes.

  Heterodimer and antigen binding polypeptide constructs and heterodimer pairs are generally purified to substantially homogeneity. The phrases “substantially homogeneous”, “substantially homogeneous form” and “substantially homogeneous” are substantially devoid of by-products resulting from a combination of polypeptides where the product is undesirable (eg, homodimers). Used to indicate that In this context, the species of interest is a heterodimer comprising H1 and L1 (H1-L1) or H2 and L2 (H2-L2). Contaminants include heterodimers containing H1 and L2 (H1-L2) or H2 and L1 (H2-L1), or homodimers containing H1 and L1 or H2 and L2 (the Fab part is precisely Included, whether paired or mispaired). Expressed in terms of purity, substantial homogeneity means that the amount of by-products does not exceed 10% of the total LC-MS intensity of all species present in the mixture, for example, less than 5%, less than 1% or 0.5% The percentage reflects the results of mass spectrometry.

  The phrase “selectively (or specifically) hybridizes” means that a particular nucleotide sequence exists only in a complex mixture (including but not limited to whole cell or library DNA or RNA). Sometimes, it means to bind, replicate or hybridize molecules under stringent hybridization conditions.

  Terms understood by those skilled in the art of antibody technology, respectively, present meanings obtained in the art, unless specifically defined differently herein. Antibodies are known to have a variable region, a hinge region and a constant domain. The structure and function of immunoglobulins is reviewed, for example, in Harlow et al., Ed., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

  As used herein, the terms “antibody” and “immunoglobulin” or “antigen-binding polypeptide construct” are used interchangeably. An “antigen-binding polypeptide construct” refers to a polypeptide that is substantially encoded by an immunoglobulin gene or one or more fragments thereof and that specifically binds to an analyte (antigen). The recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant domain genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and then define immunoglobulin isotypes IgG, IgM, IgA, IgD, and IgE, respectively. Furthermore, antibodies can belong to one of a number of subtypes, for example, IgG can belong to an IgG1, IgG2, IgG3 or IgG4 subclass.

  An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair comprising one “light” chain (about 25 kD) and one “heavy” chain (about 50-70 kD). Have. The term “light chain” includes full-length light chains and fragments thereof having a variable region sequence sufficient to confer binding specificity. The full-length light chain includes a variable region domain VL and a constant region domain CL. The variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains. The term “heavy chain” includes full-length heavy chains and fragments thereof having a variable region sequence sufficient to confer binding specificity. The full length heavy chain includes a variable region domain VH and three constant region domains CH1, CH2 and CH3. The VH domain is at the amino terminus of the polypeptide, the CH domain is at the carboxy terminus, and CH3 is closest to the carboxy terminus of the polypeptide. The heavy chain can be of any isotype including IgG (including IgG1, IgG2, IgG3 and IgG4 subclasses), IgA (including IgA1 and IgA2 subclasses), IgM, and IgE. The term “variable region” or “variable domain” typically comprises approximately 120-130 amino terminal amino acids in the heavy chain (VH) and about 100-110 amino terminal amino acids in the light chain (VL). , Generally refers to the portion of the light and / or heavy chain of an antibody that is involved in antigen recognition. A “complementarity determining region” or “CDR” is an amino acid sequence that contributes to antigen binding properties and affinity. The “framework” region (FR) can help maintain the correct conformation of the CDRs and facilitate binding of the antigen binding region to the antigen. Structurally, framework regions can be located between CDRs in an antibody. A variable region typically exhibits the same general structure, with a relatively protected framework region (FR) combined with three hypervariable region CDRs. The CDRs of each pair of two strands are typically aligned by a framework region, which allows binding to a specific epitope. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The designation of amino acids in each domain is typically in accordance with the definition of Kabat Sequences of Proteins of Immunological Intersect (National Institutes of Health, Bethesda, Md. (1987 and 1991)). In certain embodiments, the antigen binding polypeptide construct comprises at least one immunoglobulin domain of IgG, IgM, IgA, IgD, or IgE that is therapeutic polypeptide bound. In some embodiments, the immunoglobulin domain included in the antigen binding polypeptide construct provided herein is of an immunoglobulin-based construct, such as a diabody or nanobody. In certain embodiments, an antigen-binding polypeptide construct described herein comprises at least one immunoglobulin domain of a heavy chain antibody, such as a camel antibody. In certain embodiments, antigen-binding polypeptide constructs provided herein include bovine antibodies, human antibodies, camel antibodies (single domain and non-single domain), rodent antibodies, humanized antibodies, It comprises at least one immunoglobulin domain from a mammalian antibody, such as a non-humanized antibody, a murine antibody or any chimeric antibody. In certain embodiments, the antigen binding polypeptide constructs provided herein comprise at least one immunoglobulin domain of an antibody generated from a synthetic library.

  A “bi-specific”, “dual-specific” or “bifunctional” antigen binding protein or antibody is a hybrid antigen having two different antigen binding sites. It is a binding protein. Bispecific antigen binding proteins and antibodies are a type of multispecific antigen binding protein antibody. The two binding sites of the bispecific antigen binding protein and the antibody bind to two different epitopes that may be present on the same or different molecular targets. A “multispecific antigen binding protein” or “multispecific antibody” targets more than one antigen or epitope. A “bivalent antigen binding protein” or “bivalent antibody” comprises two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. Bivalent antigen binding proteins and bivalent antibodies can be bispecific. See above. It is understood that bivalent antibodies other than “multispecific” or “multifunctional” antibodies typically have the same binding site, respectively, in certain embodiments.

  The term “preferential pairing” is used herein to describe the pairing pattern of a first polypeptide and a second polypeptide, eg, antigen binding as described herein. An immunoglobulin heavy chain and an immunoglobulin light chain in polypeptide constructs and heterodimers. Thus, “preferential pairing” refers to the first polymorphism when one or more additional different polypeptides are present simultaneously when pairing occurs between the first and second polypeptides. Refers to preferential pairing between a peptide and a second polypeptide. Typically, preferential pairing occurs as a result of modifications (eg, amino acid modifications) of one or both of the first and second polypeptides. Typically, preferential pairing results in the paired first and second polypeptides being the most abundant dimer after pairing occurs. The immunoglobulin heavy chain (H1), when co-expressed with two different immunoglobulin light chains (L1 and L2), is paired statistically equally with both light chains and paired with H1 and L2 paired with L1. It is known in the art to produce an approximately 50:50 mixture of combined H1. In this context, “preferential pairing” means, for example, when H1 is coexpressed with L1 and L2, the amount of H1-L1 heavy chain-light chain heterodimer is greater than that of H1-L2 heterodimer. If greater than the amount, it occurs between H1 and L1. Therefore, in this case, H1 is preferentially paired with L1 over L2.

  However, in the context of wild-type bispecific antibodies generated from two starting antibody systems, the essential bias that the light chain of one antibody system preferentially pairs with the heavy chain of both antibody systems is It is also known to exist in some cases. Thus, when determining the strength of a design in the context of a bispecific antibody binding construct, it may be necessary to evaluate the degree of design pairing compared to the degree of wild-type pairing. Thus, in one embodiment, the design takes into account preferential matching when the amount of desired bispecific antibody is greater than the amount of desired bispecific antibody obtained in the wild-type system. Is done. In another embodiment, the design is considered to show preferential pairing if the amount of pairing in the weak arm of the antibody is greater than that found in the wild type system.

  The heavy chains of the antibody pair with the light chains of the antibody and meet or contact each other at one or more “interfaces”. “Interface” includes one or more “contact” amino acids of a first polypeptide that interact with one or more “contact” amino acid residues of a second polypeptide. For example, an interface exists between the CH3 polypeptide sequence of the dimerized CH3 domain, between the CH1 domain of the heavy chain and the CL domain of the light chain, and between the VH domain of the heavy chain and the VL domain of the light chain. The “interface” can be derived from an IgG antibody, eg, a human IgG1 antibody.

  The term “amino acid modification” as used herein includes, but is not limited to, amino acid mutations, insertions, deletions, substitutions, chemical modifications, physical modifications and rearrangements.

Antigen Binding Polypeptide Constructs and Heterodimer Pairs The antigen binding polypeptide constructs described herein can comprise a first heterodimer and a second heterodimer, each heterozygous Dimers are obtained by pairing immunoglobulin heavy chains with immunoglobulin light chains. The structure and organization of immunoglobulin heavy and light chain constant and variable domains are well known in the art. An immunoglobulin heavy chain typically comprises one variable (VH) domain and three constant domains, CH1, CH2 and CH3. An immunoglobulin light chain typically comprises one variable (VL) domain and one constant (CL) domain. Various modifications can be made to these typical formats.

  The antigen-binding polypeptide constructs and heterodimer pairs described herein can include a first heterodimer and a second heterodimer, each heterodimer comprising: An immunoglobulin / antibody heavy chain comprising at least the VH and CH1 domains or fragments thereof, and an immunoglobulin / antibody light chain having a VL domain and a CL domain. In one embodiment, the heterodimer of both the heterodimer pair and the antigen binding polypeptide construct comprises a full-length immunoglobulin heavy chain. In another embodiment, the heterodimer of both the heterodimer pair or antigen-binding polypeptide construct comprises a fragment of an immunoglobulin heavy chain comprising at least the VH and CH1 domains. In one embodiment, both heterodimers of a heterodimer pair comprise an amino terminal fragment of an immunoglobulin heavy chain comprising at least the VH and CH1 domains. In another embodiment, both heterodimers of a heterodimer pair comprise a carboxy terminal fragment of an immunoglobulin heavy chain comprising at least the VH and CH1 domains.

  Each heterodimer of a heterodimer pair can specifically bind to an antigen or epitope. In one embodiment, each heterodimeric immunoglobulin heavy chain and immunoglobulin light chain is derived or modified from a known antibody, eg, a therapeutic antibody. A therapeutic antibody is effective in treating a disease or disorder in a mammal that has or is susceptible to a disease or disorder. Suitable therapeutic antibodies from which the respective heterodimers can be derived include abagovomab (adagomomab), adalimumab, alemtuzumab, aurograb, bapineuzumab, basiliximab, berimumab, bevacizumab, briakinumumab, canakitumumab, canakitumumab Gol, cetuximab, daclizumab, denosumab, efalizumab, gariximab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, rumilizimab, mepolizumab, motolizumab, motabizumab ), Ocrelizumab, ofatumumab, omalizumab, palivizumab, panits Mab, pertuzumab, ranibizumab, Resurizumabu, rituximab, Tepurizumabu, tocilizumab / atlizumab (Atlizumab), tositumomab, trastuzumab, Proxinium (TM), Rencarex (TM) include, but are Usutekinumabu and zalutumumab, but are not limited to.

  In one embodiment, each heterodimeric immunoglobulin heavy chain and / or immunoglobulin light chain comprises the following list of proteins and subunits, domains, motifs and epitopes belonging to the following list of proteins: Derived or modified from an antibody that binds to, but is not limited to, a molecule. Renin; growth hormone including human and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; Hormones; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VII, factor VIIIC, factor IX, tissue factor (TF) and von Willebrand factor; coagulation factors such as protein C; atrial natriuretic factor : Pulmonary surfactant; plasminogen activator such as urokinase or human urine or tissue plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor-alpha and beta; enkephalinase R NTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); serum such as human serum albumin Albumin; Muller tube inhibitor; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin related peptide; microbial protein such as beta-lactamase; DNase; IgE; cytotoxic T lymphocyte related antigen such as CTLA-4 (CTLA); inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptors such as EGFR, VEGFR; -Interferons such as ferron (alpha-IFN), beta interferon (beta-IFN) and gamma interferon (gamma-IFN); protein A or D; rheumatoid factor; bone derived neurotrophic factor (BDNF), neurotrophin-3,- Neurotrophic factors such as 4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factor; platelet derived growth factor (PDGF); fibroblasts such as AFGF and PFGF Cell growth factor; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta including TGF-1, TGF-2, TGF-3, TGF-4 or TGF-5; insulin -Like growth factors I and II (IGF-I and IGF-II); Death (1-3) -IG -I (brain IGF-I), insulin-like growth factor binding protein; CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD40, CD40L, CD52, CD proteins such as CD63, CD64, CD80 and CD147; erythropoietin; bone inducing factor; immunotoxin; bone morphogenetic protein (BMP); interferons such as interferon alpha, beta and gamma; M-CSF, GM-CSF and G-CSF, etc. Colony-stimulating factor (CSF); interleukins (ILs) such as IL-1 to IL-13; TNF-alpha, superoxide dismutase; T cell receptor; surface membrane protein; Viral antigens such as gp120; transport proteins; homing receptors; addressins; regulatory proteins; cells such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM A4 / p7 integrin and (Xv / p3 integrin; CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, alpha7, alpha8, alpha9, alphaD, CD11a, CD11b, CD51 , Integrin alpha subunits such as CD11c, CD41, alpha IIb, alpha IELb; integrin beta subunits such as CD29, CD18, CD61, CD104, beta 5, beta 6, beta 7 and beta 8 A combination of integrin subunits including but not limited to alpha Vbeta3, alphaVbeta5 and alpha4beta7; members of the apoptotic pathway; IgE; blood group antigen; flk2 / flt3 receptor; obesity (OB ) Receptor; mpI receptor; CTLA-4; protein C; Eph receptors such as EphA2, EphA4, EphB2, etc .; human leukocyte antigens (HLA) such as HLA-DR; complements such as complement receptors CR1, C1Rq Proteins and other complement factors such as C3 and C5; glycoprotein receptors such as GpIb alpha, GPIIb / IIIa and CD200; and fragments of any of the polypeptides listed above.

  In certain embodiments, each heterodimeric immunoglobulin heavy chain and / or immunoglobulin light chain comprises an ALK receptor (pleiotrophin receptor), pleiotrophin, KS1 / 4 pancreatic cancer antigen (pan- ovarian cancer antigen (CA125); prostate acid phosphatase; prostate specific antigen (PSA); melanoma associated antigen p97; melanoma antigen gp75; high molecular melanoma antigen (HMW-MAA); prostate specific membrane antigen Carcinoembryonic antigen (CEA); polymorphic epithelial mucin antigen; human milk fat globule antigen; colorectal cancer-related antigens such as CEA, TAG-72, CO17-1A, GICA19-9, CTA-1 and LEA; Lymphoma antigen-38.13; CD19; human B lymphoma antigen CD20; CD33; gangliosi Melanoma-specific antigens such as GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor-specific transplanted cell surface antigen (TSTA); virus-induced tumor antigens including RNA tumor virus T antigen, DNA tumor virus and envelope antigen Colonic CEA, 514 carcinoembryonic trophoblast glycoprotein and bladder tumor antigen and other carcinoembryonic antigen alphafetoprotein; differentiation antigens such as human lung antigens L6 and L20; fibrosarcoma antigens; human leukemia T cell antigen Gp37; Neoglycoprotein; sphingolipid; breast cancer antigens such as EGFR (epidermal growth factor receptor); NY-BR-16; NY-BR-16 and HER2 antigen (p185HER2); polymorphic epithelial mucin (PEM); Antigen APO-1; found in fetal erythrocytes Differentiation antigens such as I antigen; early endoderm I antigen found in adult erythrocytes; preimplantation embryo; I (Ma) found in gastric adenocarcinoma; M18, M39 found in breast epithelium; SSEA found in bone marrow cells 1; VEP8; VEP9; Myl; Va4-D5; D156-22 found in colorectal cancer; TRA-1-85 (blood group H); SCP-1 found in testis and ovarian cancer; found in rectal adenocarcinoma C14; F3 found in lung adenocarcinoma; AH6 found in gastric cancer; Y hapten; Ley found in embryonic cancer cells; TL5 (blood group A); EGF receptor found in A431 cells; E1 series found in pancreatic cancer (Blood group B); FC10.2 found in embryonic cancer cells; gastric adenocarcinoma antigen; CO-514 found in adenocarcinoma (blood group Lea); NS-10 found in adenocarcinoma; CO 43 (blood group Leb); G49 found in the EGF receptor of A431 cells; MH2 found in colon adenocarcinoma (blood group ALeb / Ley); 19.9 found in colon cancer; gastric cancer mucin; found in bone marrow cells T5A7; R24 found in melanoma; 4.2 found in embryonic cancer cells, GD3, D1.1, OFA-1, GM2, OFA-2, GD2 and M1: 22: 25: 8, and embryonic 4 SSEA-3 and SSEA-4 found in ~ 8 cell stage; cutaneous T cell lymphoma antigen; MART-1 antigen; Siary Tn (STn) antigen; colon cancer antigen NY-CO-45; lung cancer antigen NY-LU-12 variant A; Adenocarcinoma antigen ART1; Paraneoplastic related brain testicular cancer antigen (oncural neuronal antigen MA2, paraneoplastic neuron antigen (p neuro-neurological abdominal antigen 2 (Neuro-oncologic ventricular antigen 2) (NOVA2); hepatocellular carcinoma antigen gene 520; tumor-associated antigen CO-029; tumor-associated antigen MAGE-C1 (cancer / testis antigen) CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4-a, MAGE-4-b and MAGE-X2; cancer-testis antigen NY-EOS-1) As well as fragments of any of the polypeptides listed above, but are derived or modified from antibodies that specifically bind to a cancer antigen.

  Human antibodies can be divided into isotypes including IgG, IgA, IgE, IgM and IgD. In one embodiment, the Fc is derived from an IgG isotype. In another embodiment, the Fc is derived from an IgA isotype. In another embodiment, the Fc is derived from an IgE isotype. In another embodiment, the Fc is derived from an IgM isotype. In another embodiment, the Fc is derived from an IgD isotype.

  Human IgG antibodies can also be divided into subclasses IgG1, IgG2, IgG3 and IgG4. Thus, in some embodiments, it is contemplated that Fc can be derived from the IgG1, IgG2, IgG3 and IgG4 subclasses of antibodies.

  Each heterodimer of a heterodimer pair can specifically bind to an epitope or antigen. In one embodiment, each heterodimer of a heterodimer pair binds to the same epitope. In another embodiment, the first heterodimer of the heterodimer pair specifically binds to an epitope of one antigen, and the second heterodimer of the heterodimer pair is the same antigen. Specifically bind to different epitopes. In another embodiment, the first heterodimer of the heterodimer pair specifically binds to an epitope of the first antigen, and the second heterodimer of the heterodimer pair is It specifically binds to an epitope of a second antigen that is different from one antigen. For example, in one embodiment, the first heterodimer specifically binds to tissue factor, while the second heterodimer specifically binds to the antigen Her2 (ErbB2), and The reverse is also true. In an alternative embodiment, the first heterodimer specifically binds to tissue factor, while the second heterodimer specifically binds to EGFR and vice versa. . In yet another embodiment, the first heterodimer specifically binds to EGFR, while the second heterodimer specifically binds to antigen Her2 and vice versa. . In another embodiment, the first heterodimer specifically binds to a molecule or cancer antigen described above. In another embodiment, the second heterodimer specifically binds to a molecule or cancer antigen described above.

  As indicated above, in some embodiments, each heterodimeric immunoglobulin heavy chain and immunoglobulin light chain binds from a known therapeutic antibody or to various target molecules or cancer antigens. May be derived from antibodies or genetically engineered from them. The amino acid and nucleotide sequences of many such molecules are readily available (eg, GenBank: AJ308808. 1 (humanized anti-human tissue factor antibody D3H44 light chain variable region and CL domain); GenBank: AJ308808.1 (Humanized anti-human tissue factor antibody D3H44 heavy chain variable region and CH1 domain); GenBank: HC359902.1 (Pertuzumab Fab light chain gene module); GenBank: HC35904.1 (Pertuzumab Fab heavy chain gene module); GenBank: GM6865465. 1 (antibody trastuzumab (= Herceptin) -wild type; light chain); GenBank: GM68563.1 (antibody trastuzumab (= Herceptin) -wild type; heavy chain); GenBank: M68546.1 (antibody trastuzumab (= Herceptin) -GC optimized light chain); and GenBank: GM68544.1 (refer to antibody trastuzumab (= Herceptin) -GC optimized heavy chain), as described herein. The respective GenBank number sequences are available from the NCBI website dated 28 November 2012 and are hereby incorporated by reference in their entirety for all purposes, including the amino acid and nucleotide sequences of cetuximab. Known in the art, for example, Canadian Institutes of Health Research, Alberta Innovates-Health Solutions, and The Metabolisms Inno. ation Centre (TMIC) by assisted Drug Bank web site, referring to the accession number DB00002.

  In some embodiments, the isolated antigen binding polypeptide construct has at least 80, 85, 90, 91, 92 amino acid sequences or fragments thereof as set forth in the tables or accession numbers disclosed herein. , 93, 94, 95, 96, 97, 98, 99 or 100% identical amino acid sequences. In some embodiments, the isolated antigen-binding polypeptide construct is at least 80, 85, 90, 91, 92 to the nucleotide sequence or fragment thereof set forth in the tables or accession numbers disclosed herein. 93, 94, 95, 96, 97, 98, 99, or 100% identical amino acid sequences encoded by the polynucleotide.

Amino acid modifications to immunoglobulin heavy and light chains At least one heterodimer of a heterodimer pair is one in which the heavy chain of the first heterodimer preferentially pairs with one light chain. One or more amino acid modifications are included in the immunoglobulin heavy chain and / or immunoglobulin light chain so as not to pair with the other. Similarly, the heavy chain of the second heterodimer can preferentially pair with the second light chain rather than the first. Preferential pairing of one heavy chain with one of two light chains is based on a design set (called LCCA design set) that includes one immunoglobulin heavy chain and two immunoglobulin light chains The immunoglobulin heavy chain preferentially pairs with one of the two immunoglobulin light chains when the immunoglobulin heavy chain is co-expressed with both immunoglobulin light chains. Thus, the LCCA design set can include one immunoglobulin heavy chain, a first immunoglobulin light chain, and a second immunoglobulin light chain.

  In one embodiment, the one or more amino acid modifications include one or more amino acid substitutions.

  In one embodiment, the preferential pairing demonstrated by the LCCA design set is established by modifying one or more amino acids that are part of the interface between the light and heavy chains. In one embodiment, the preferential pairing demonstrated by the LCCA design set is the CH1 domain of an immunoglobulin heavy chain, the CL domain of a first immunoglobulin light chain and the CL domain of a second immunoglobulin light chain. Established by modifying one or more amino acids in at least one.

  In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains as indicated by the residue Kabat numbering. Has been. For example, Almagro [Frontiers In Bioscience (2008) 13: 1619-1633] provides a definition of framework residues based on Kabat, Chotia and IMGT numbering.

  In one embodiment, at least one of the heterodimers comprises one or more mutations introduced into an immunoglobulin heavy chain and an immunoglobulin light chain that are complementary to each other. Complementarity at the heavy and light chain interfaces can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces is documented in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc. It has been widely described and all include the conformity of structural and chemical properties between two interacting surfaces. In one embodiment, at least one of the heterodimers has a mutation introduced into an immunoglobulin heavy chain and an immunoglobulin light chain that introduces a new hydrogen bond into the heavy and light chains at the interface. Or contains multiple mutations. In one embodiment, at least one of the heterodimers has a mutation introduced into an immunoglobulin heavy chain and an immunoglobulin light chain that introduces a new salt bridge to the heavy and light chains at the interface. Or contains multiple mutations.

  Non-limiting examples of suitable LCCA design sets are described in the examples, tables and figures. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least one amino acid modification in the CH1 domain, a first immunoglobulin light chain having at least one amino acid modification in the CL domain, and an amino acid in the CL domain. Includes a second immunoglobulin light chain with no modifications. In another embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least one amino acid modification in the CH1 domain, a first immunoglobulin light chain having at least one amino acid modification in the CL domain, and at least in the CL domain. It includes a second immunoglobulin light chain with one amino acid modification. In another embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least one amino acid modification in the CH1 domain, a first immunoglobulin light chain having at least two amino acid modifications in the CL domain, and at least in the CL domain. A second immunoglobulin light chain with two amino acid modifications is included. In another embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least one amino acid modification in the CH1 domain, a first immunoglobulin light chain having at least two amino acid modifications in the CL domain, and at least in the CL domain. It includes a second immunoglobulin light chain with one amino acid modification.

  In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having no amino acid modification in the CH1 domain, a first immunoglobulin light chain having no amino acid modification in the CL domain, and at least one amino acid in the CL domain. A second immunoglobulin light chain having a modification is included. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain that has no amino acid modification in the CH1 domain, a first immunoglobulin light chain that has no amino acid modification in the CL domain, and at least two amino acids in the CL domain. A second immunoglobulin light chain having a modification is included.

  In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least two amino acid modifications in the CH1 domain, a first immunoglobulin light chain having no amino acid modification in the CL domain, and at least one in the CL domain. A second immunoglobulin light chain having the following amino acid modifications: In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least two amino acid modifications in the CH1 domain, a first immunoglobulin light chain having at least one amino acid modification in the CL domain, and at least in the CL domain. It includes a second immunoglobulin light chain with one amino acid modification. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least two amino acid modifications in the CH1 domain, a first immunoglobulin light chain having at least one amino acid modification in the CL domain, and at least in the CL domain. A second immunoglobulin light chain with two amino acid modifications is included. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 2 amino acid modifications in the CH1 domain, a first immunoglobulin light chain having at least 2 amino acid modifications in the CL domain, and at least in the CL domain. A second immunoglobulin light chain with two amino acid modifications is included. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 2 amino acid modifications in the CH1 domain, a first immunoglobulin light chain having at least 3 amino acid modifications in the CL domain, and at least in the CL domain. A second immunoglobulin light chain with two amino acid modifications is included.

  In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 3 amino acid modifications in the CH1 domain, a first immunoglobulin light chain having no amino acid modification in the CL domain, and at least one in the CL domain. A second immunoglobulin light chain having the following amino acid modifications: In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 3 amino acid modifications in the CH1 domain, a first immunoglobulin light chain having at least one amino acid modification in the CL domain, and at least in the CL domain. It includes a second immunoglobulin light chain with one amino acid modification. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 3 amino acid modifications in the CH1 domain, a first immunoglobulin light chain having at least 3 amino acid modifications in the CL domain, and at least in the CL domain. A second immunoglobulin light chain with two amino acid modifications is included. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 3 amino acid modifications in the CH1 domain, a first immunoglobulin light chain having at least 4 amino acid modifications in the CL domain, and at least in the CL domain. It includes a second immunoglobulin light chain with three amino acid modifications. In one embodiment, the preferential pairing demonstrated by the LCCA design set is a VH domain of an immunoglobulin heavy chain, a VL domain of a first immunoglobulin light chain, and a VL domain of a second immunoglobulin light chain. Established by modifying one or more amino acids in at least one. Non-limiting examples of suitable LCCA design sets are shown in the table and examples below.

  In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain without an amino acid modification in the VH domain, a first immunoglobulin light chain without an amino acid modification in the VL domain, and at least one amino acid in the VL domain. A second immunoglobulin light chain having a modification is included. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain without amino acid modification in the VH domain, a first immunoglobulin light chain without amino acid modification in the VL domain, and at least two amino acids in the VL domain. A second immunoglobulin light chain having a modification is included.

  In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least one amino acid modification in the VH domain, a first immunoglobulin light chain having no amino acid modification in the VL domain, and at least one in the VL domain. A second immunoglobulin light chain having the following amino acid modifications: In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least one amino acid modification in the VH domain, a first immunoglobulin light chain having at least one amino acid modification in the VL domain, and at least in the VL domain. It includes a second immunoglobulin light chain with one amino acid modification. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least one amino acid modification in the VH domain, a first immunoglobulin light chain having at least two amino acid modifications in the VL domain, and at least in the VL domain. A second immunoglobulin light chain with two amino acid modifications is included.

  In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least two amino acid modifications in the VH domain, a first immunoglobulin light chain having no amino acid modification in the VL domain, and at least one in the VL domain. A second immunoglobulin light chain having the following amino acid modifications: In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 2 amino acid modifications in the VH domain, a first immunoglobulin light chain having at least 2 amino acid modifications in the VL domain, and at least in the VL domain. It includes a second immunoglobulin light chain with one amino acid modification. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least two amino acid modifications in the VH domain, a first immunoglobulin light chain having at least one amino acid modification in the VL domain, and at least in the VL domain. It includes a second immunoglobulin light chain with one amino acid modification.

  In one embodiment, the LCCA design set is combined to provide a combination comprising two separate immunoglobulin heavy chains (H1 and H2) and two separate immunoglobulin light chains (L1 and L2), wherein When H1, H2, L1 and L2 are co-expressed, H1 preferentially pairs with L1, and H2 preferentially binds with L2.

  In some embodiments, the amino acid modifications described herein are in the context of a bispecific antibody construct. For example, the design set described herein can be incorporated into a full-length immunoglobulin heavy chain such that the full-length heavy chain preferentially pairs with an immunoglobulin light chain. In some embodiments, the full length immunoglobulin heavy chain contains amino acid modifications that promote dimerization in the Fc region, as described in the Examples.

Possibility of transfer of specific amino acid modifications specified herein to other antibodies:
Specific amino acid modifications to the immunoglobulin heavy and light chains identified above have been described for immunoglobulin heavy and light chains of D3H44 anti-tissue factor extracellular domain antibodies, trastuzumab and cetuximab, but these amino acids The modifications can be transferred to other immunoglobulin heavy chains and light chains, resulting in a similar pattern of preferential pairing of one immunoglobulin heavy chain and one of the two immunoglobulin light chains as follows: Are considered and demonstrated herein (see, eg, Examples, Figures and Tables).

  VH: VL and CH1: CL interface residues at the immunoglobulin heavy and light chain interface are relatively well conserved (Padlan et al., 1986, Mol. Immunol. 23 (9): 951-960). . This sequence conservation is the result of evolutionary suppression and increases the likelihood that a functionally active antigen binding domain will be formed upon the combined pairing of light and heavy chains. As a result of this sequence conservation, the sequence modifications in the specific examples described above for D3H44 that lead to preferential pairings are transferred to other heavy and light chain pair heterodimers to give approximately equal preferential pairings. Can be obtained, since this region shows high sequence conservation throughout the antibody. Furthermore, if sequence differences occur, these are usually distal from the CH1: CL interface. This is especially true in the CH1 and CL domains. However, with respect to CDR (complementarity determining region) loop residues (and length), particularly CDR-H3, there is some sequence variability in the antigen binding site. Thus, in one embodiment, the heterodimer pairs described herein are such that at least one heterodimer has one or more when the amino acid sequence of the antigen binding site is significantly different from the D3H44 antibody. A heterodimer comprising the amino acid modification of VH and / or VL domains distal to the CDR loop. In another embodiment, the heterodimer pair described herein has at least one heterodimer when the amino acid sequence of the antigen binding site is substantially the same as the D3H44 antibody, or Includes heterodimers that include multiple amino acid modifications in the VH and / or VL domains proximal or distal to the CDR loop.

  In one embodiment, the amino acid modifications described herein can be transferred to immunoglobulin heavy and light chains of human or humanized IgG1 / κ based antibodies. Non-limiting examples of such IgG1 / κ chains include ofatumumab (human) or trastuzumab, pertuzumab or bevacizumab (humanized).

  In another embodiment, the amino acid modifications described herein are transferable to immunoglobulin heavy and light chains of antibodies utilizing commonly used VH and VL subgroups. A non-limiting example of such an antibody includes pertuzumab.

  In one embodiment, the amino acid modifications described herein can be transferred to immunoglobulin heavy and light chains of antibodies having a framework close to the germline. An example of such an antibody includes obinutuzumab.

  In one embodiment, the amino acid modifications described herein are of the immunoglobulin heavy and light chain of an antibody having a VH: VL interdomain angle close to that observed on average for heavy and light chain pairs. Can be transferred to a chain. An example of this type of antibody includes, but is not limited to, pertuzumab. In another embodiment, the amino acid modifications described herein are transferable to immunoglobulin heavy and light chains of antibodies having basic CL and CH1 domains. Suitable examples of such antibodies include, but are not limited to trastuzumab.

  In some embodiments, a particular subset of the amino acid modifications described herein are utilized for the variable domains of the antigen binding constructs provided above.

  Examples, Figures and Tables show that amino acid modifications (eg, within one or more Fab fragments including variable and constant regions) can be transferred to other immunoglobulin heavy and light chains, one immunoglobulin It has been demonstrated that it results in a similar pattern of preferential pairing of the heavy chain with one of the two immunoglobulin light chains.

Preferential Pairing As described above, at least one heterodimer of the antigen binding construct / heterodimer pairs described herein is a heavy chain of one heterodimer, For example, H1 preferentially pairs with one of the light chains, eg L1, and not the other light chain L2, and the other heterodimeric heavy chain H2 preferentially with light chain L2. One or more amino acid modifications may be included in the immunoglobulin heavy chain and / or the immunoglobulin light chain so that they will pair with each other and not with the light chain L1. In other words, it is considered that the desired preferential pairing is between H1 and L1 and between H2 and L2. For example, the preferential pairing of H1 and L1 is that when H1 is combined with L1 and L2, the yield of H1-L1 heterodimer is relative to the H2 / L2 pair without one or more amino acid modifications. , For each pairing of the corresponding H1 / L1 pair, it is considered to occur if it is greater than the yield of the mispaired H1-L2 heterodimer. Similarly, the preferential pairing of H2 and L2 is that when H2 is combined with L1 and L2, the yield of H2-L2 heterodimer is H2 / L2 pair without one or more amino acid modifications. For each pair of corresponding H1 / L1 pairs is considered to occur when it is greater than the yield of mispaired H2-L1 heterodimers. In this context, heterodimers comprising H1 and L1 (H1-L1) or H2 and L2 (H2-L2) are preferentially paired, precisely paired, absolute pairs or desired While heterodimers comprising H1 and L2 (H1-L2) or H2 and L1 (H2-L1) are referred to herein as mismatched heterodimers. Called in the calligraphy. A set of mutations corresponding to two heavy chains and two light chains that are intended to achieve selective pairing of H1-L1 and H2-L2 is called a design set.

  Thus, in one embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the relative yield of the desired heterodimer is greater than 55%. In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the relative yield of the desired heterodimer is greater than 60%. In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the relative yield of the desired heterodimer is greater than 70%. In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the relative yield of the desired heterodimer is greater than 80%. In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the relative yield of the desired heterodimer is greater than 90%. In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the relative yield of the desired heterodimer is greater than 95%.

  In the above example, preferential pairing of H1-L1 is that when the amount of the desired H1-L1 heterodimer is such that when H1 is coexpressed with L1 and L2, the mispaired H1-L2 heterodimer It is considered to occur when it is larger than the amount. Similarly, preferential pairing of H2-L2 is greater than the amount of mispaired H2-L2 heterodimers when the amount of the desired H2-L2 heterodimer is coexpressed with H1 and L2. Is also considered to occur when it is large. Thus, in one embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the ratio of desired heterodimer to mismatched heterodimer is: It exceeds 1.25: 1. In one embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the ratio of desired heterodimer to mismatched heterodimer is: Over 5: 1. In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the ratio of desired heterodimer to mismatched heterodimer is 2: Over 1 In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the ratio of desired heterodimer to mismatched heterodimer is 3: Over 1 In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the ratio of desired heterodimer to mismatched heterodimer is 5: Over 1 In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the ratio of desired heterodimer to mismatched heterodimer is 10: Over 1 In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the ratio of desired heterodimer to mismatched heterodimer is 25: Over 1 In another embodiment, when one immunoglobulin heavy chain of a heterodimer is coexpressed with two immunoglobulin light chains, the ratio of desired heterodimer to mismatched heterodimer is 50: 1. Over.

  In some embodiments, the heterodimers described herein preferentially pair to form a bispecific antibody. In some embodiments, the construct comprises a heterodimer that preferentially pairs to form a bispecific antibody selected from D3H44 / trastuzumab, D3H44 / cetuximab and trastuzumab / cetuximab. In some embodiments, bispecific antibodies comprise the amino acid modifications described in Tables 28a-28c.

  In some embodiments, two full length heavy chain constructs are co-expressed with two unique light chain constructs to generate 10 possible antibody species: H1-L1: H1-L1, H1-L2: H1. -L2, H1-L1: H1-L2, H2-L1: H2-L1, H2-L2: H2-L2, H2-L1: H2-L2, H1-L1: H2-L1, H1-L2: H2-L2 , H1-L2: H2-L1 and H1-L1: H2-L2. The H1-L1: H2-L2 species are considered to be correctly paired bispecific antibody species. In some embodiments, the DNA ratio is selected to yield the maximum amount of precisely paired bispecific antibody species. For example, in some embodiments, the ratio of H1: H2: L1: L2 is 15: 15: 53: 17. In some embodiments, the ratio of H1: H2: L1: L2 is 15: 15: 17: 53.

  In some embodiments, the percentage of correctly paired bispecific species is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 compared to all species. %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (eg, Tables 29a-29c and 30a-30c). In some embodiments, the percentage of correctly paired bispecific species co-express the corresponding wild type H1, H2, L1, and L2 without the amino acid modifications described in Tables 28a-28c. Greater than the percentage of correctly paired bispecific antibody species obtained. In some embodiments, the percentage of correctly paired bispecific species co-express the corresponding wild type H1, H2, L1, and L2 without the amino acid modifications described in Tables 28a-28c. At least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70% compared to the percentage of precisely matched bispecific antibody species obtained by Or 75% increase.

Heterodimer Thermal Stability In addition to facilitating preferential pairing, amino acid substitutions were chosen so that the mutation did not destabilize the Fab heterodimer. Thus, in most cases, Fab heterodimer stability measurements were very close to wild-type Fab (eg, within 3 ° C. of wild-type Fab).

  Thus, in some embodiments, each heterodimer of a heterodimer pair described herein comprises the same immunoglobulin heavy chain and light chain, but is described herein. Has thermal stability comparable to heterodimers without amino acid modifications in the CH1, CL, VH or VL domains. In one embodiment, thermal stability is determined by measuring melting temperature or Tm. Thus, in one embodiment, the thermostability of the heterodimer described herein comprises the same immunoglobulin heavy chain and light chain but as described herein CH1, CL, VH or Within about 10 ° C. of heterodimer with no amino acid modification in the VL domain. Accordingly, in one embodiment, the thermostability of the heterodimer described herein includes the same immunoglobulin heavy and light chains but is described herein as CH1, CL, VH. Or within about 5 ° C. of a heterodimer with no amino acid modification in the VL domain. In another embodiment, the thermostability of the heterodimer described herein comprises the same immunoglobulin heavy chain and light chain but as described herein CH1, CL, VH or VL. Within about 3 ° C. of heterodimer with no amino acid modifications in the domain. In another embodiment, the thermostability of the heterodimer described herein comprises the same immunoglobulin heavy chain and light chain but as described herein CH1, CL, VH or VL. Within about 2 ° C. of heterodimer with no amino acid modifications in the domain. In another embodiment, the thermostability of the heterodimer described herein comprises the same immunoglobulin heavy chain and light chain but as described herein CH1, CL, VH or VL. Within about 1.5 ° C. of heterodimer with no amino acid modification in the domain. In another embodiment, the thermostability of the heterodimer described herein comprises the same immunoglobulin heavy chain and light chain but as described herein CH1, CL, VH or VL. Within about 1 ° C. of heterodimer with no amino acid modifications in the domain. In another embodiment, the thermostability of the heterodimer described herein comprises the same immunoglobulin heavy chain and light chain but as described herein CH1, CL, VH or VL. Within about 0.5 ° C. of heterodimer with no amino acid modifications in the domain. In another embodiment, the thermostability of the heterodimer described herein comprises the same immunoglobulin heavy chain and light chain but as described herein CH1, CL, VH or VL. Within about 0.25 ° C. of heterodimer with no amino acid modifications in the domain.

  Further, in some embodiments, the thermostability of the heterodimer described herein comprises the same immunoglobulin heavy and light chains but is described herein as CH1, CL, Surprisingly improved (ie increased) compared to heterodimers that do not have amino acid modifications in the VH or VL domains. Thus, in one embodiment, the thermostability of the heterodimer described herein comprises CH1, CL, VH or the same immunoglobulin heavy chain and light chain, but described herein. About 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 compared to heterodimers without amino acid modifications in the VL domain 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2 .1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7 3.8, 3.9, 4.0, 4.5, 5.0 ° C. or more.

Affinity of Heterodimer to Antigen In one embodiment, each heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but is described herein. Have the same or comparable affinity for the respective antigen as the heterodimer without amino acid modifications in the CL, VH or VL domains. In one embodiment, a heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but has amino acid modifications in the CH1, CL, VH or VL domains described herein. Each antigen has an affinity within about 50 times that of the non-heterodimer. In one embodiment, a heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but has amino acid modifications in the CH1, CL, VH or VL domains described herein. Each antigen has an affinity within about 25 times that of the heterodimer that does not. In one embodiment, a heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but has amino acid modifications in the CH1, CL, VH or VL domains described herein. Each antigen has an affinity within about 10 times that of the heterodimer that does not. In another embodiment, the heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but has amino acid modifications in the CH1, CL, VH or VL domains described herein. Each antigen has an affinity within about 5 times that of the heterodimer that does not. In another embodiment, the heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but has amino acid modifications in the CH1, CL, VH or VL domains described herein. Each antigen has an affinity within about 2.5 times that of the non-heterodimer. In another embodiment, the heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but has amino acid modifications in the CH1, CL, VH or VL domains described herein. Each antigen has an affinity within about twice that of the heterodimer that does not. In another embodiment, the heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but has amino acid modifications in the CH1, CL, VH or VL domains described herein. Each antigen has an affinity within about 1.5 times that of the heterodimer that does not. In another embodiment, the heterodimer of a heterodimer pair comprises the same immunoglobulin heavy and light chains but has amino acid modifications in the CH1, CL, VH or VL domains described herein. Each antigen has approximately the same affinity as the heterodimer that does not.

Additional Selective Modifications In one embodiment, the heterodimer pairs of immunoglobulin heavy and light chains described herein are covalently linked to prevent preferential pairing of heavy and light chains. Further modification (ie, by covalent attachment of various types of molecules) so as not to affect the ability of the heterodimer to bind to the antigen or affect the stability of the heterodimer can do. Such modifications include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Including, but not limited to. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

  In another embodiment, the heterodimer pairs of immunoglobulin heavy and light chains described herein are attached (directly or indirectly) to a portion of a therapeutic agent or drug that modifies a given biological response. Can be conjugated). The therapeutic agent or drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Such proteins include, for example, a toxin, such as abrin, ricin A, Onconase (or another cytotoxic RNase), Pseudomonas exotoxin, cholera toxin or diphtheria toxin; tumor necrosis factor, alpha interferon, beta Interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, apoptotic agent such as TNF-alpha, TNF-beta, AIM I (see WO 97/33899), AIM II (See WO 97/34911), Fas ligand (Takahashi et al., 1994, J. Immunol., 6: 1567) and VEGI (see WO 99/23105), thrombus-forming agent Or anti-blood Angiogenic agents, eg, proteins such as angiostatin or endostatin; or, for example, lymphokines (eg, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin- 6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF") and granulocyte colony stimulating factor ("G-CSF")), or growth factors (e.g. , Growth hormone ("GH")).

  Further, in alternative embodiments, the antibody can be conjugated to a therapeutic moiety such as a radioactive material or macrocyclic chelator useful for conjugating a radioactive metal ion (for examples of radioactive materials). See above). In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ″ -tetraacetic acid (which can be attached to the antibody via a linker molecule. DOTA). Such linker molecules are generally known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4: 2483; Peterson et al., 1999, Bioconjug. Chem. 10: 553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26: 943.

  In some embodiments, the heterodimeric immunoglobulin heavy and light chains are expressed as a fusion protein that includes a tag to facilitate purification and / or testing and the like. As referred to herein, a “tag” is any appended series of amino acids that are provided to a protein either at the C-terminus, N-terminus, or internally and contribute to protein identification or purification. Those skilled in the art know that suitable tags are useful for the purification and / or testing of albumin binding domains (ABD), His tags, FLAG tags, glutathione-s-transferases, hemagglutinin (HA) and maltose binding proteins. But are not limited to these. Such tagged proteins can also be modified to include cleavage sites such as thrombin, enterokinase or factor X cleavage sites for easy removal of tags before, during or after purification.

  In some embodiments, one of the cysteine residues below the Fab domain in the light chain (position 214, Kabat numbering) and heavy chain (position 233, Kabat numbering) that form an interchain disulfide bond, or Multiple can be modified to serine, or alanine, or non-cysteine, or another amino acid.

  It is contemplated that additional amino acid modifications can be made to the immunoglobulin heavy chain to increase the level of preferential pairing and / or thermal stability of the heterodimer pair. For example, additional amino acid modifications can be made to the immunoglobulin heavy chain Fc domain, leading to preferential pairings for heterodimer pairs compared to homodimer pairs. Such amino acid modifications are known in the art and include, for example, those described in US Patent Publication No. 2012/0149876. Alternatively, for example, “knob into hole”, charged residues with ionic interactions and strand exchange modified domain (SEED) technology, etc. Alternative strategies that lead to matching can be used. The latter strategy is described in the art and reviewed in Klein et al., Supra. Further discussion on Fc domains follows below.

Fc domain In embodiments where the antigen binding polypeptide construct comprises a full length immunoglobulin heavy chain, the construct comprises an Fc. In some embodiments, the Fc comprises at least one or two CH3 domain sequences. In some embodiments, when the antigen-binding polypeptide construct comprises a heterodimer that includes only the Fab region of the heavy chain, the Fc is the first with or without one or more linkers. It binds to the heterodimer and / or the second heterodimer. In some embodiments, the Fc is a human Fc. In some embodiments, the Fc is IgG or IgG1 Fc. In some embodiments, the Fc is a heterodimeric Fc. In some embodiments, the Fc comprises at least one or two CH2 domain sequences.

In some embodiments, the Fc comprises one or more modifications in at least one of the _CH3 domain sequences. In some embodiments, the Fc comprises one or more modifications in at least one of the _CH2 domain sequences. In some embodiments, the Fc is a single polypeptide. In some embodiments, the Fc is a plurality of polypeptides, eg, two polypeptides.

In some embodiments, the Fc comprises one or more modifications in at least one of the _CH3 sequences. In some embodiments, the Fc comprises one or more modifications in at least one of the _CH2 sequences. In some embodiments, the Fc is a single polypeptide. In some embodiments, the Fc is a plurality of polypeptides, eg, two polypeptides.

  In some embodiments, the Fc is the Fc described in the PCT / CA2011 / 001238 filed November 4, 2011 and the PCT / CA2012 / 050780 patent application filed November 2, 2012, these The entire disclosure of each is hereby incorporated by reference in its entirety for all purposes.

  In some embodiments, the constructs described herein comprise a heterodimeric Fc comprising a modified CH3 domain that is asymmetrically modified. A heterodimeric Fc can comprise two heavy chain constant domain polypeptides: a first heavy chain polypeptide and a second heavy chain polypeptide, which can be used interchangeably, provided that The condition is that the Fc comprises one first heavy chain polypeptide and one second heavy chain polypeptide. In general, the first heavy chain polypeptide comprises a first CH3 sequence and the second heavy chain polypeptide comprises a second CH3 sequence.

  Two CH3 sequences containing one or more amino acid modifications introduced asymmetrically generally result in heterodimers Fc rather than homodimers when the two CH3 sequences dimerize. As used herein, an “asymmetric amino acid modification” refers to an amino acid at a particular position in a first CH3 sequence that differs from an amino acid in a second CH3 sequence at the same position, and the first and second CH3 sequences Refers to any modification that preferentially pairs to form a heterodimer rather than a homodimer. This heterodimerization is a modification of only one of the two amino acids at the same corresponding amino acid position in each sequence, or the respective sequence at the same corresponding position in each of the first and second CH3 sequences. May be the result of modification of both amino acids. The first and second CH3 sequences of the heterodimeric Fc can include one or more than one asymmetric amino acid modification.

  Table X provides the amino acid sequence of the human IgG1 Fc sequence corresponding to amino acids 231 to 447 of the full length human IgG1 heavy chain. The CH3 sequence includes amino acids 341-447 of the full length human IgG1 heavy chain.

  Typically, an Fc can include two consecutive heavy chain sequences (A and B) that can be dimerized. In some embodiments, one or both sequences of Fc, using EU numbering, are one or the following in the following positions: L351, F405, Y407, T366, K392, T394, T350, S400 and / or N390 Contains multiple mutations or modifications. In some embodiments, the Fc comprises a mutant sequence shown in Table X. In some embodiments, the Fc comprises a variant 1A-B mutation. In some embodiments, the Fc comprises a variant 2A-B mutation. In some embodiments, the Fc comprises a variant 3A-B mutation. In some embodiments, the Fc comprises a variant 4A-B mutation. In some embodiments, the Fc comprises a variant 5A-B mutation.

(Table X)

  The first and second CH3 sequences can include amino acid mutations described herein with reference to amino acids 231 to 447 of the full-length human IgG1 heavy chain. In one embodiment, the heterodimeric Fc comprises a modified CH3 domain in which the first CH3 sequence has amino acid modifications at positions F405 and Y407, and the second CH3 sequence has an amino acid modification at position T394. In one embodiment, the heterodimeric Fc has one or more amino acid modifications wherein the first CH3 sequence is selected from L351Y, F405A and Y407V, and the second CH3 sequence is T366L, T366I, K392L. A modified CH3 domain having one or more amino acid modifications selected from K392M and T394W.

  In one embodiment, the heterodimer Fc has a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407, and a second CH3 sequence having amino acid modifications at positions T366, K392 and T394. , One of the first or second CH3 sequences comprises a modified CH3 domain further comprising an amino acid modification at position Q347 and the other CH3 sequence further comprises an amino acid modification at position K360. In another embodiment, the heterodimeric Fc has a first CH3 sequence with amino acid modifications at positions L351, F405 and Y407, and a second CH3 sequence has amino acid modifications at positions T366, K392 and T394. , One of the first or second CH3 sequences further comprises an amino acid modification at position Q347, the other CH3 sequence further comprises an amino acid modification at position K360, wherein one or both of said CH3 sequences comprises the amino acid modification T350V In addition, it contains a modified CH3 domain.

  In one embodiment, the heterodimer Fc has a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407, and a second CH3 sequence having amino acid modifications at positions T366, K392 and T394. A modification wherein one of the first or second CH3 sequences further comprises an amino acid modification of D399R or D399K, and the other CH3 sequence comprises one or more of T411E, T411D, K409E, K409D, K392E and K392D Contains the CH3 domain. In another embodiment, the heterodimeric Fc has a first CH3 sequence with amino acid modifications at positions L351, F405 and Y407, and a second CH3 sequence has amino acid modifications at positions T366, K392 and T394. Wherein one of the first or second CH3 sequences further comprises an amino acid modification of D399R or D399K, and the other CH3 sequence comprises one or more of T411E, T411D, K409E, K409D, K392E and K392D, One or both of the CH3 sequences comprises a modified CH3 domain that further comprises the amino acid modification T350V.

  In one embodiment, the heterodimer Fc has a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407 and a second CH3 sequence having amino acid modifications at positions T366, K392 and T394. , Wherein one or both of said CH3 sequences further comprises a modified CH3 domain further comprising an amino acid modification of T350V.

  In one embodiment, the heterodimeric Fc comprises a modified CH3 domain comprising the following amino acid modification, wherein “A” comprises an amino acid modification to the first CH3 sequence and “B” is: Contains an amino acid modification to the second CH3 sequence. A: L351Y_F405A_Y407V, B: T366L_K392M_T394W, A: L351Y_F405A_Y407V, B: T366L_K392L_T394W, A: T350V_L351Y_F405A_Y407V, B: T350V_T366L_K392L_T394W, A: T350V_L351Y_F405A_Y407V, B: T350V_T366L_K392M_T394W, A: T350V_L351Y_S400E_F405A_Y407V and / or B: T350V_T366L_N390R_K392M_T394W.

One or more asymmetric amino acid modifications can promote the formation of a heterodimeric Fc in which the heterodimeric CH3 domain has comparable stability to the wild type homodimeric CH3 domain. In certain embodiments, the one or more asymmetric amino acid modifications promote the formation of a heterodimeric Fc domain where the heterodimeric Fc domain has a stability comparable to a wild type homodimeric Fc domain. In certain embodiments, the one or more asymmetric amino acid modifications are such that the heterodimeric Fc domain is stable as observed by melting temperature (Tm) in a differential scanning calorimetry study, with a corresponding melting temperature. Promotes formation of heterodimeric Fc domains that are within 4 ° C. of the melting temperature observed in symmetric wild type homodimeric Fc domains. In some embodiments, the Fc comprises one or more modifications in at least one of the _CH3 sequences that promotes the formation of a heterodimeric Fc with stability comparable to the wild-type homodimeric Fc. .

In one embodiment, the stability of the CH3 domain can be assessed, for example, by measuring the melting temperature of the CH3 domain by differential scanning calorimetry (DSC). Thus, in a further embodiment, the CH3 domain has a melting temperature of about 68 ° C. or higher. In another embodiment, the CH3 domain has a melting temperature of about 70 ° C. or higher. In another embodiment, the CH3 domain has a melting temperature of about 72 ° C. or higher. In another embodiment, the CH3 domain has a melting temperature of about 73 ° C. or higher. In another embodiment, the CH3 domain has a melting temperature of about 75 ° C. or higher. In another embodiment, the CH3 domain has a melting temperature of about 78 ° C. or higher. In some embodiments, the dimerized C _H3 sequence is about 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, It has a melting temperature (Tm) of 83, 84 or 85 ° C or higher.

  In some embodiments, a heterodimeric Fc comprising a modified CH3 sequence can be formed with a purity of at least about 75% as compared to a homodimeric Fc in the expression product. In another embodiment, the heterodimeric Fc is formed with a purity greater than about 80%. In another embodiment, the heterodimeric Fc is formed with a purity greater than about 85%. In another embodiment, the heterodimeric Fc is formed with a purity greater than about 90%. In another embodiment, the heterodimeric Fc is formed with a purity greater than about 95%. In another embodiment, the heterodimeric Fc is formed with a purity greater than about 97%. In some embodiments, the Fc, when expressed, is about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92. , 93, 94, 95, 96, 97, 98, or a heterodimer formed with a purity greater than 99%. In some embodiments, the Fc is about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 when expressed through a single cell. , 90, 91, 92, 93, 94, 95, 96, 97, 98 or a purity of greater than 99% heterodimer.

  Additional methods for modifying monomeric Fc polypeptides to promote heterodimeric Fc formation are described in International Patent Publication No. WO 96/027011 (Knob Into Hall), Gunasekaran et al. (Gunasekaran K. et al. (2010). J Biol Chem. 285, 19637-46, electrostatic design to achive selective heterodimation), Davis et al. (Davis, JH. Et al. (2010) Prot Eng Des sel; 23 (4): e g technology) and Labrijn et al. [Efficient generation of stable]. e bispecific IgG1 by controlled Fab-arm exchange. Labrijn AF, Meesters JI, de Goeij BE, van den Bremer ET, Neijssen J, van Kampen MD, Strumane K, Verplogen S, Kundu A, Gramer MJv. Proc Natl Acad Sci USA. 2013 Mar 26; 110 (13): 5145-50. In some embodiments, the isolated constructs described herein are more stable and more productive than antigen binding constructs that bind to an antigen and antigen binding constructs that do not include the same Fc polypeptide. Includes dimeric Fc polypeptide constructs with superior biophysical characteristics such as ease. Numerous mutations in the heavy chain sequence of Fc are known in the art to selectively alter the affinity of antibody Fc for different Fc gamma receptors. In some embodiments, the Fc comprises one or more modifications that facilitate selective binding of the Fc gamma receptor.

  The CH2 domain is amino acids 231 to 340 of the sequence shown in Table X. Exemplary mutations are listed below.

  S298A / E333A / K334A, S298A / E333A / K334A / K326A (Lu Y, Vernes JM, Chiang N, et al. J Immunol Methods. 2011 Feb 28; 365 (1-2): 132/41); V305I / P396L, F243L / R292P / Y300L / L235V / P396L (Stavenhagen JB, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep 15; 67 (18): 8882-90; Breast Cancer Res. 2011 Nov 30; 13 (6): R123); F243L (Stewa t R, Thom G, Leaves M, et al. Protein Eng Des Sel. 2011 Sep; 24 (9): 671-8.), S298A / E333A / K334A (Shields RL, Namenuk AK, Hong K, et al. Jl. Biol. Mar 2; 276 (9): 6591-604); S239D / I332E / A330L, S239D / I332E (Lazar GA, Dang W, Karki S, et al. Proc Natl Acad Sci USA. 2006 Mar 14; 103 (11): 4005- 10); S239D / S267E, S267E / L328F (Chu SY, Vostial I, Karki S, et al. Mol Immunol. 2008 Sep; 45 (15): 39. 6-33); S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I362S / L3302E / / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D, and other mutations listed in WO2011 / 120134 and WO2011 / 120135, which are incorporated herein by reference. Therapeutic Antibiotic Engineering (William R. Strohl and Lila M. Strohl, Woodpublishing series in Biomedicine No 11, ISBN 1 907568 t 20

  In some embodiments, the CH2 domain comprises one or more asymmetric amino acid modifications. In some embodiments, the CH2 domain comprises one or more asymmetric amino acid modifications that promote selective binding of FcγR. In some embodiments, the CH2 domain allows for separation and purification of the isolated constructs described herein.

FcRn binding and PK parameters As known in the art, binding to FcRn recirculates endocytosed antibodies from endosomes into the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181). -220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766). This process, combined with hindrance of renal filtration due to the large size of the full length molecule, results in a favorable antibody serum half-life ranging from 1 to 3 weeks. Fc binding to FcRn also plays a major role in antibody transport. Thus, in one embodiment, the constructs of the invention can bind to FcRn.

Additional modifications to improve effector function In some embodiments, the constructs described herein can be modified to improve effector function. Such modifications are known in the art and are directed to non-fucosylation, or activated receptors, mainly directed to FCGR3a for ADCC and directed to C1q for CDC Genetic engineering of the affinity of the Fc portion of Table Y below summarizes the various designs reported in the literature for effector function operations.

(Table Y)

  Accordingly, in one embodiment, a construct described herein comprises a dimeric Fc comprising one or more amino acid modifications shown in the table above that confer improved effector function. Can do. In another embodiment, the construct can be nonfucosylated to improve effector function.

Linkers The constructs described herein can include one or more heterodimers described herein operably linked to an Fc described herein. . In some embodiments, the Fc binds to one or more heterodimers with or without one or more linkers. In some embodiments, the Fc binds directly to one or more heterodimers. In some embodiments, the Fc is linked to one or more heterodimers by one or more linkers. In some embodiments, the Fc is linked to the heavy chain of each heterodimer by a linker.

  In some embodiments, the one or more linkers are one or more polypeptide linkers. In some embodiments, the one or more linkers comprise one or more antibody hinge regions. In some embodiments, the one or more linkers comprise one or more IgG1 hinge regions.

Methods for Preparing Dimer Pairs As described above, the heterodimer pairs described herein can include a first heterodimer and a second heterodimer. Each heterodimer includes an immunoglobulin heavy chain or fragment thereof comprising at least the VH and CH1 domains, and an immunoglobulin light chain having a VL domain and a CL domain. Heterodimeric immunoglobulin heavy chains and immunoglobulin light chains can be readily prepared using recombinant DNA techniques known in the art. For example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd Edition, 2001); , Cold Spring Harbor, NY, 2nd edition, 1989); Short Protocols in Molecular Biology (Ausubel et al., John Wiley and Sons, New York, 4th edition, 1999); and GlickP as ak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, DC, 2nd edition, 1998) using standard techniques such as recombinant nucleic acid method, nucleic acid synthesis, It can be used for cell culture, transgene integration and recombinant protein expression. Alternatively, the heterodimers and heterodimer pairs described herein can be chemically synthesized.

  The immunoglobulin heavy and light chain nucleic acid and amino acid sequences of the antibody from which the heterodimer is derived are known in the art or can be readily determined by use of nucleic acid and / or protein sequencing methods. it can. Methods for gene fusion of the tags described herein to immunoglobulin heavy chains and / or light chains are known in the art and some are described below and in the Examples.

  For example, methods for expressing and coexpressing immunoglobulin heavy and light chains in a host are well known in the art. In addition, methods for tagging heavy and / or light chains using recombinant DNA techniques are well known in the art. Expression vectors and hosts suitable for the expression of heavy and light chains are also well known in the art and are described below.

  Bispecific antibody production methods that do not rely on the use of only single clones or transient cell lines that express all four chains are known in the art (Gramer, et al. (2013) mAbs 5 962, Strop et al. (2012) J Mol Biol 420, 204). These methods rely on post-production arm exchange under redox conditions of two pairs of light and heavy chains involved in the formation of bispecific antibodies (redox production). In this approach, the H1: L1 and H2: L2 pairs can be expressed in two different cell lines to produce two Fab arms independently. Subsequently, the two Fab arms are mixed under selective redox conditions to achieve reassociation of the two unique heavy chains H1 and H2, resulting in dual specificity including L1: H1: H2: L2 chains Form antibodies. It may be envisaged to use the library / dataset of the design described herein in the production of double antibodies using the redox production method or a variation of that method.

  In certain embodiments, a cell-free protein expression system is utilized to co-express polypeptides (eg, polypeptide heavy and light chains) without the use of living cells. Rather, all components necessary to transcribe DNA into RNA and transcribe RNA into proteins (eg, ribosomes, tRNAs, enzymes, cofactors, amino acids) are in solution used in vitro. Provided. In certain embodiments, in vitro expression requires (1) a gene template (mRNA or DNA) encoding heavy and light chain polypeptides, and (2) a reaction solution containing the necessary transcriptional and translational molecular mechanisms. And In certain embodiments, the cell extract contains components of the reaction solution, e.g., RNA polymerase for mRNA transcription, ribosomes for polypeptide translation, tRNA, amino acids, enzyme cofactors, energy sources and accurate Essentially supplies the essential cellular components for protein folding. A cell-free protein expression system can be prepared using lysates obtained from bacterial cells, yeast cells, insect cells, plant cells, mammalian cells, human cells or combinations thereof. Such cell lysates can provide the exact composition and proportion of enzymes and components required for translation. In some embodiments, the cell membrane is removed, leaving only the cell cytoplasm and organelle components.

  Several cell-free protein expression systems are described in Carlson et al. (2012) Biotechnol. Adv. 30: 1185-1194, as known in the art. For example, cell-free protein expression systems based on prokaryotic cells or eukaryotic cells can be used. Examples of a prokaryotic cell expression system include those of E. coli. Examples of eukaryotic cell expression systems that can be used are, for example, those based on extracts of rabbit reticulocytes, wheat germ and insect cells. Such prokaryotic or eukaryotic protein expression systems are commercially available from companies such as Roche, Invitrogen, Qiagen and Novagen. One skilled in the art can readily select a cell-free protein expression system suitable for producing polypeptides that can pair with each other (eg, heavy and light chain polypeptides). In addition, cell-free protein expression systems can be supplemented with chaperones (eg, BiP) and isomerases (eg, disulfide isomerase) to improve the efficiency of IgG folding.

  In some embodiments, a cell-free expression system is utilized to co-express heavy and light chain polypeptides from a DNA template (transcription and translation) or an mRNA template (translation only).

Vectors and host cells Recombinant expression of heavy and light chains requires the construction of expression vectors containing polynucleotides encoding heavy or light chains (eg, antibodies or fusion proteins). Once a polynucleotide encoding a heavy or light chain is obtained, vectors for producing heavy or light chains can be produced by recombinant DNA techniques using techniques well known in the art. Accordingly, methods for preparing a protein by expressing a polynucleotide containing a heavy or light chain encoding a nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing heavy or light chain coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. The invention thus provides a replicable vector comprising a nucleotide sequence encoding a heavy or light chain operably linked to a promoter.

  The expression vector is transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the modified heavy or light chain used in the methods of the invention. In certain embodiments, the heavy and light chains used in the method are co-expressed in a host cell for expression of the entire immunoglobulin molecule and are detailed below.

  A variety of host expression vector systems may be utilized to express the modified heavy and light chains. Such host expression systems represent vehicles in which the coding sequence of interest can be produced and subsequently purified, and when transformed or transfected with the appropriate nucleotide coding sequence, the modified heavy and light chains are converted into their Also represents cells that can be expressed in situ. These include microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing modified heavy and light chain coding sequences; Yeast transformed with expression vectors for recombinant yeast containing modified heavy and light chain coding sequences (eg, Saccharomyces, Pichia); recombination containing modified heavy and light chain coding sequences Insect cell lines infected with viral expression vectors (eg, baculovirus); Recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco zykevirus, TMV) or modified heavy and light chain coding Recombination containing sequences A plant cell line transformed with a lasmid expression vector (eg, Ti plasmid); or a promoter derived from the genome of a mammalian cell (eg, a metallothionein promoter) or a promoter derived from a mammalian virus (eg, an adenovirus late promoter) Mammalian cell lines (eg, COS, CHO, BHK, HEK-293, NSO, and 3T3 cells) with a recombinant expression construct containing a vaccinia virus 7.5K promoter), but are not limited to these. In certain embodiments, bacterial cells or eukaryotic cells such as Escherichia coli are used for the expression of modified heavy and light chains that are recombinant antibodies or fusion protein molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO), together with vectors such as human cytomegalovirus major intermediate early gene promoter elements, are effective expression systems for antibodies (Foecking et al., 1986). Gene 45: 101; and Cockett et al., 1990, Bio / Technology 8: 2). In certain embodiments, the expression of nucleotide sequences encoding each heterodimeric immunoglobulin heavy chain and light chain is regulated by a constitutive, inducible or tissue specific promoter.

  In mammalian host cells, a number of viral-based expression systems are available. In cases where an adenovirus is used as an expression vector, the desired modified heavy and light chain coding sequences can be ligated to an adenovirus transcription / translation control complex, eg, the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, region E1 or E3) results in a recombinant virus in which expression of modified heavy and light chains in an infected host is practical (Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 355-359). A specific initiation signal may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon must be aligned with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bittner et al., 1987, Methods in Enzymol. 153: 516-544).

  Heterodimeric immunoglobulin heavy and light chain expression can be controlled by any promoter or enhancer element known in the art. Promoters that can be used to control the expression of genes encoding modified heavy and light chains (eg, antibodies or fusion proteins) include the SV40 early promoter region (Bernist and Chamber, 1981, Nature 290: 304-310), Rous Promoter contained in the 3 'long terminal repeat of sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. US A. 78.1441-1445), regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42), tetracycline (Tet) promoter (Gossen et al., 1995, Pr). oc.Nat.Acad.Sci.USA89: 5547-5551); β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731) or the tac promoter (See also DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25; “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94). Prokaryotic expression vectors of: nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303: 209-213) or cauliflower mosaic A plant expression vector comprising the viral 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9: 2871) and the promoter of the photosynthetic enzyme ribulose diphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310: 115-120); Yeast or other fungal promoter elements such as Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animals showing tissue specificity and used as transgenic animals Transcriptional regulatory region: Elastase I gene regulatory region active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639) -646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515), insulin gene control region active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122), immunoglobulin gene control active in lymphocytes Regions (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444), ovary, breast, lymph and Mouse mammary tumor virus regulatory region active in mast cells (Leder et al., 1986, Cell 45: 485-495), albumin gene regulatory region active in liver (Pinke) rt et al., 1987, Genes and Devel. 1: 268-276), alpha-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science. 235: 53-58), alpha 1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), beta-globin gene regulatory region active in bone marrow cells (Mogram et al. 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94), myelin basic protein gene system active in brain oligodendrocyte cells. Region (Readhead et al., 1987, Cell 48: 703-712), myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314: 283-286), neuron-specific active in neurons Enolase (NSE) (Morelli et al., 1999, Gen. Virol. 80: 571-83), brain-derived neurotrophic factor (BDNF) gene regulatory region active in neurons (Tabuchi et al., 1998, Biochem. Biophysic. Res. Com. 253: 818-823), glial fibrillary acidic protein (GFAP) promoter active in astrocytes (Gomes et al., 1999, Braz J Med Biol Res 32 (5): 619-631; Morelli et al., 999, Gen. Virol. 80: 571-83) and the gonadotropin-releasing hormone gene control region active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

  In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific desired fashion. Expression by a specific promoter can be increased in the presence of a specific inducer, thereby controlling the expression of the genetically modified fusion protein. Furthermore, different host cells have characteristic and specific mechanisms in translation and post-translational processing and modification (eg, protein glycosylation, phosphorylation). Appropriate cell lines or host systems are chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system produces a non-glycosylated product, and expression in yeast produces a glycosylated product. Eukaryotic host cells with cellular mechanisms (eg, glycosylation and phosphorylation) that accurately process the primary transcript of the gene product can be used. Such mammalian host cells include CHO, VERY, BHK, Hela, COS, MDCK, HEK-293, 3T3, WI38, NSO, in particular, for example, SK-N-AS, SK-N-FI, SK-N. -DZ human neuroblastoma (Sugimoto et al., 1984, J. Natl. Cancer Inst. 73: 51-57), SK-N-SH human neuroblastoma (Biochim. Biophys. Acta, 1982, 704: 450- 460), Daoy human cerebellar medulloblastoma (He et al., 1992, Cancer Res. 52: 1144-1148), DBTRG-05MG glioblastoma cells (Kruse et al., 1992, In Vitro Cell. Dev. Biol. 28A: 609-614), IMR-32 human neuroblastoma (Ca cer Res., 1970, 30: 2110-2118), 1321 N1 human astrocytoma (Proc. Natl. Acad. Sci. USA, 1977, 74: 4816), MOG-G-CCM human astrocytoma (Br) J. Cancer, 1984, 49: 269), U87MG human glioblastoma-astrocytoma (Acta Pathol. Microbiol. Scand., 1968, 74: 465-486), A172 human glioblastoma (Olopede et al., 1992). Cancer Res. 52: 2523-2529), C6 rat glioma cells (Benda et al., 1968, Science 161: 370-371), Neuro-2a mouse neuroblastoma (Proc. Natl. Acad. Sci. USA, 1970, 6 129-136), NB41A3 mouse neuroblastoma (Proc. Natl. Acad. Sci. USA, 1962, 48: 1184-1190), SCP sheep choroid plexus (Bolin et al., 1994, J. Virol. Methods 48: 211). -221), G355-5, PG-4 feline normal astrocytes (Haapala et al., 1985, J. Virol. 53: 827-833), Mpf ferret brain (Trowbridge et al., 1982, In Vitro 18: 952-960). ) And normal cells such as CTX TNA2 rat normal cerebral cortex (Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6467-6471), eg CRL7030 and Hs578Bst. Including but vesicular systems, without limitation. In addition, different vector / host expression systems can affect processing reactions to different extents.

  Stable expression is often preferred for long term, high yield production of recombinant proteins. For example, cell lines that stably express the modified heavy and light chains (eg, antibodies or fusion proteins) of the invention can be engineered. Instead of using an expression vector containing a viral origin of replication, the host cell can be transformed into DNA and selective markers controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) Can be used to transform. After the introduction of exogenous DNA, the modified cells were grown for 1-2 days in rich medium and then replaced with selective medium. Selectable markers in recombinant plasmids confer resistance to selection, allowing cells to stably integrate the plasmid into the chromosome and propagate to form growth foci, which are then cloned into cell lines. Can be propagated.

  A number of selection systems can be used, including herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA. 48: 2026) and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can be used for, but not limited to, tk-, hgprt- or aprt-cells, respectively. It also provides resistance to methotrexate as a selection criterion for the dhfr gene (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA. 78: 1527), confer resistance to mycophenolic acid as a selection criterion for the gpt gene (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072), aminoglycoside G- as a selection criterion for the neo gene Confer resistance to 418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1) and confer resistance to hygromycin as a selection criterion for the hygro gene (Sante re et, 1984, Gene 30: 147), can be used antimetabolite resistance.

Co-expression of heavy and light chains The heterodimeric immunoglobulin heavy and light chains described herein can be co-expressed in mammalian cells, as indicated above. In one embodiment, one heavy chain is coexpressed with two different light chains of the LCCA design set described above, and the heavy chain preferentially pairs with one of the two light chains. In another embodiment, two unique heavy chains are co-expressed with two unique light chains, each heavy chain preferentially pairing with one of the light chains.

Heterodimer Pair Testing As described above, at least one heterodimer of the heterodimer pairs described herein is one of the two unique weights of the heterodimer pair. When a chain and two unique light chains are co-expressed in mammalian cells, the first heterodimeric heavy chain preferentially pairs with one light chain but not the other. One or more amino acid modifications can be included in the immunoglobulin heavy chain and / or immunoglobulin light chain. Similarly, the heavy chain of the second heterodimer preferentially pairs with the second light chain rather than the first. The degree of preferential pairing can be assessed using, for example, the methods described below. The affinity of each heterodimer of the heterodimer pair to the corresponding antigen can be tested as described below. The thermal stability of each heterodimer of the heterodimer pair can also be tested as described below.

Precedence measurement method LCCA
In one embodiment, the preferential pairing of immunoglobulin heavy and light chains is determined by performing a light chain competition assay (LCCA). Shared patent application PCT / US2013 / 063306, filed Oct. 3, 2013, describes various embodiments of LCCA, which is incorporated herein by reference in its entirety for all purposes. The method allows quantitative analysis of heavy and specific light chain pairings within a mixture of co-expressed proteins, where one specific immunoglobulin heavy chain is two when the heavy and light chains are co-expressed. It can be used to determine whether it selectively associates with either immunoglobulin light chain. The method is briefly described below. At least one heavy chain and two different light chains are co-expressed in the cell in a ratio such that the heavy chain is a limited pair of reaction pairs, optionally separating the secreted protein from the cell and binding to the heavy chain Separating the immunoglobulin light chain polypeptide from the remainder of the secreted protein to produce isolated heavy chain paired fractions, detecting the amount of each different light chain in the isolated heavy chain fraction; The relative amount of each different light chain in the isolated heavy chain fraction is analyzed to determine the ability of at least one heavy chain to selectively pair with one light chain.

  The method provides reasonable throughput, is robust (ie, less sensitive to minor changes in performance, such as user or flow rate), and is accurate. The method provides a sensitive assay that can measure the effects of small variations in protein sequence. Irregular protein-protein, domain-domain, chain-chain interactions over a large area usually require multiple mutations (replacements) to introduce selectivity. There is no need to isolate and purify the protein product, which allows for more efficient sorting. Further details regarding this method embodiment are described in the Examples.

Alternative Methods for Determining Preferential Pairing Alternative methods for detecting preferential pairing use molecular weight differences to identify each distinct species, each a relative heterodimer containing a light chain. Use of LC-MS (Liquid Chromatography / Mass Spectrometry) to quantify the population. Antigen activity assays can also be used to quantify relative heterodimeric populations, each containing a light chain, using the degree of binding measured (compared to the control), each corresponding Estimate heterodimeric populations to

  Additional methods such as SMCA are described in the examples, figures and tables.

Thermal Stability The thermal stability of the heterodimer can be determined according to methods known in the art. The melting temperature of each heterodimer indicates thermal stability. The melting point of the heterodimer can be determined using techniques such as differential scanning calorimetry (Chen et al. (2003) Pharm Res 20: 1952-60; Ghirlando et al. (1999) Immunol Lett 68: 47-52). ). Alternatively, the thermal stability of the heterodimer can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40: 343-9).

Affinity to antigen The binding affinity of the heterodimer to the corresponding antigen and the off-rate of the interaction can be determined by competitive binding assays according to methods well known in the art. One example of a competitive binding assay includes incubating a labeled antigen (eg, 3H or 125I) and a molecule of interest (eg, a heterodimer of the invention) in the presence of increasing amounts of unlabeled antigen and Detecting a molecule bound to the labeled ligand. The affinity of the heterodimer of the present invention for antigen and the rate of binding off can be determined from saturation data by Scatchard analysis.

  The heterodimer kinetic parameters described herein can also be determined using surface plasmon resonance (SPR) based assays known in the art (eg, BIAcore kinetic analysis). it can. Review articles on SPR-based techniques include Mullet et al., 2000, Methods 22: 77-91; Dong et al., 2002, Review in Mol. Biotech. 82: 303-23; Fivas et al., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al., 2000, Current Opinion in Biotechnology 11: 54-61. Additionally, described in US Pat. Nos. 6,373,577, 6,289,286, 5,322,798, 5,341,215, and 6,268,125 Both SPR instruments and SPR-based methods for measuring protein-protein interactions that are being considered are contemplated by the methods of the present invention. FACS can also be used to measure affinity and is known in the art.

A library of bispecific antibody mutation design sets is used to generate predetermined Mabl and Mab2 of bispecific antibodies. In one embodiment, two bases comprising antigen binding fragments Fab1 and Fab2, respectively, and Described herein are bispecific antibody mutation design sets aimed at selectively forming bispecific antibodies, starting from the antibodies Mab1 and Mab2. The design set consists of cognate mutations corresponding to Fab1, Fab2 and Fc, respectively. In one embodiment, the design set library is represented by a design set included in any of Table 5, Table 12, or Tables 15-17. Mutations are introduced at the Fab1 light and heavy chain interface to achieve selective pairing of the two absolute chains in the presence of Fab2 competing light and heavy chains. Selective pairing directs preferred complementary mutations into two absolute light and heavy chains based on steric, hydrophobic or electrostatic complementarity between specific hot spot framework residues at the interface. While achieving these mutant residues by participating in unfavorable interfacial interactions with non-absolute chain pairs. In each design set, selective pairing mutations were introduced at the Fab2 light and heavy chain interface to select two absolute chains in the presence of Fab1 competing light and heavy chains. Target matching can also be achieved. Mutations are also aimed at reducing the mispairing of Fab1 light chain and Fab2 heavy chain and vice versa. Mutations are introduced at the Fc interface to achieve selective pairing of heavy chains to form asymmetric antibody molecules containing two different heavy chains. Genetic engineering of specific interfacial residue positions in an antibody's light and heavy chains often results in deleterious effects such as loss of the antibody's antigen binding affinity, stability, solubility, aggregation properties, etc. Can bring A number of related properties can be affected, such as kon and koff rates, melting temperature (Tm), acid, base, oxidation, freeze / thaw, agitation, stability to stress conditions such as pressure and the like. This is often affected by the complementarity determining region (CDR) of the antibody of interest. If the CDRs of different antibodies are generally not identical, the effect of the mutation design set on the properties described above may not be the same across all antibodies. A method for producing Mab1 and Mab2 of any two available antibodies of a bispecific antibody having a distinct purity compared to an incorrectly paired antibody-like structure containing other contaminants is described herein. Presented in Mab1 and Mab2 light and heavy chains are coexpressed after the introduction of cognate mutations in their respective mutation design sets, and the expressed antibody products are analytically screened to other Mab-like species expressed in protein products. In contrast, the purity of the preferred bispecific antibody is estimated. In some embodiments, the analytical sorting procedure may be based on LC-MS technology. In some embodiments, the analytical sorting procedure may be based on charge-based separation, such as capillary isoelectric focusing (cIEF) techniques or chromatographic techniques. An example of a sorting technique is presented in Example 9 based on the SMCA procedure. In some embodiments, the outstanding purity of the bispecific antibody is defined as greater than 70% of all Mab-like species obtained in the expressed protein product. In some embodiments, the outstanding purity of the bispecific antibody is defined as greater than 90% of all Mab-like species obtained in the expressed protein product. The procedure for the preparation and selection of a given Mab1 and Mab2 of the bispecific Mab design set is shown schematically in FIG.

Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising a heterodimer or heterodimer pair as described herein. Such compositions comprise a therapeutically effective amount of a heterodimer or heterodimer pair and a pharmaceutically acceptable carrier. In certain embodiments, the term “pharmaceutically acceptable” is approved by federal or state government regulatory authorities, or is recognized by the United States Pharmacopeia or other commonly accepted use for animals, particularly humans. Means that it was presented to the pharmacopoeia. The term “carrier” refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be oils, including water and petroleum, animal, vegetable or synthetic sources such as sterile liquid peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, barley, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene , Glycol, water, ethanol and the like. If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are E. W. As described in “Remington's Pharmaceutical Sciences” by Martin. Such compositions contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the correct mode of administration to the patient. The formulation should suit the mode of administration.

  In certain embodiments, a composition comprising a heterodimer or heterodimer pair is formulated according to routine procedures in pharmaceutical compositions adapted for intravenous administration to humans. Typically, intravenous administration-like compositions are solutions with sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizer and a local anesthetic such as lignocaine to ease pain at the site of the injection. In general, the ingredients are provided separately or mixed together in a unit dosage form, for example, as a lyophilized powder or anhydrous concentrate contained in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent . For compositions to be administered by infusion, they can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

  In certain embodiments, the compositions described herein are formulated in neutral or salt form. Pharmaceutically acceptable salts include anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, Those formed by cations such as those derived from triethylamine, 2-ethylaminoethanol, histidine, procaine and the like are included.

  The amount of a composition described herein that is effective in treating, inhibiting and preventing a disease or disorder associated with abnormal expression and / or activity of a therapeutic protein should be determined by standard clinical techniques. Can do. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The exact dosage used in the formulation will also depend on the route of administration and the severity of the disease or disorder and should be determined by the judgment of the physician and the circumstances of each patient. Effective doses are extrapolated from dose-response curves derived from in vitro or animal model test systems.

Use of Heterodimer Pairs As described above, the heterodimer pairs described herein can include a first heterodimer and a second heterodimer. Each heterodimeric immunoglobulin heavy chain and / or immunoglobulin light chain comprises one or more modifications from a known therapeutic antibody or from a known antibody that binds to the molecule. Accordingly, it is contemplated that heterodimers containing modifications to these antibodies can be used in the treatment or prevention of the same disease, disorder or infection for which known therapeutic antibodies or known antibodies can be used.

  In another embodiment, the heterodimer pairs described herein are combined with other therapeutic agents known in the art in the treatment or prevention of cancer, autoimmune diseases, inflammatory disorders or infections Therefore, it can be used advantageously. In certain embodiments, the heterodimeric pairs described herein are combined with monoclonal or chimeric antibodies, lymphokines, or hematopoietic growth factors such as IL-2, IL-3 and IL-7 This can be used, for example, to increase the number or activity of effectors that interact with the molecule and to increase the immune response. The heterodimeric pairs described herein are advantageously combined with one or more drugs used to treat a disease, disorder or infection, such as, for example, an anti-cancer agent, anti-inflammatory agent or anti-viral agent It can also be used.

Kits The present invention additionally provides kits that include one or more heterodimer pairs. The individual components in the kit are packaged in separate containers and can be associated with such containers with a labeling form prescribed by the administrative authority that regulates the manufacture, use or sale of a pharmaceutical or biological product. The label reflects the authorization of manufacture, use or sale by the authorities. The kit can optionally contain instructions or instructions that outline how to use the heterodimer pair or dosing regimen.

  When one or more components of the kit are provided as a solution, for example as an aqueous or sterile aqueous solution, the container means is itself an inhaler, syringe, pipette, eye drop container or other such device. From which the solution can be administered to the subject or can be applied or mixed with other components of the kit.

  The components of the kit can also be provided in a dried or lyophilized form, and the kit can additionally contain a solvent suitable for reconstitution of the lyophilized component. Regardless of the number or type of containers, the kits of the invention can also include devices to assist in administering the composition to the patient. Such devices can be inhalers, intranasal sprayers, syringes, pipettes, forceps, measuring spoons, eye drops containers or similar medically approved delivery vehicles.

Computer Implementation In one embodiment, the computer includes at least one processor coupled to the chipset. A memory, storage device, keyboard, device, graphics adapter, pointing device, and network adapter are also coupled to the chipset. The display is coupled to the graphics adapter. In one embodiment, chipset functionality is provided by a memory controller hub and an I / O controller hub. In another embodiment, the memory is directly coupled to the processor rather than the chipset.

  A storage device is any device capable of holding data, such as a hard drive, a compact disk read only memory (CD-ROM), a DVD or a semiconductor memory device. The memory holds instructions and data used by the processor. The pointing device can be a mouse, trackball or other type of pointing device and is used in combination with a keyboard to enter data into a computer system. The graphics adapter displays images and other information on the display. A network adapter couples a computer system to a local or wide area network.

  As is known in the art, a computer may have different and / or other components than those described above. In addition, the computer may lack certain components. Further, the storage device may be local and / or remote from the computer (eg, incorporated within a storage area network (SAN)).

  As is known in the art, a computer is adapted to execute computer program modules to provide the functionality described herein. As used herein, the term “module” refers to computer program logic utilized to provide a particular functionality. Thus, the module can be implemented by hardware, firmware and / or software. In one embodiment, the program module is stored in a storage device, loaded into memory, and executed by a processor.

  The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in that respect will be suggested to those skilled in the art, within the spirit and scope of this application, As well as within the scope of the appended claims.

  The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental error and deviation should, of course, be allowed for.

  The practice of the present invention employs conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art unless otherwise indicated. Such techniques are explained fully in the literature. For example, T.W. E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S.C. Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic P Rukoto.

Example 1: Molecular modeling and computer-guided manipulation of the Fab interface Heavy and light chain abrupts that can be screened in the context of other antibodies (Abs) or fragments thereof using structural and computational molecular modeling guidance techniques A library of mutation designs was generated to identify mutations that exhibit the desired specificity in the antibody of interest. A design strategy to manipulate preferential heavy chain (H) -light chain (L) pairings included first identifying a representative Fab (ie, D3H44).

  As shown in Table 1, the main criteria for this Fab are human / humanized, having commonly used VH and VL subgroups, and minimal framework region mutations. It was to contain. In addition, structural considerations were that the VH: VL interdomain angle should be close to the average observed in antibodies. After selecting Fab D3H44, computer analysis of the Fab interface was performed to identify and understand residues important for heavy and light chain interactions by using a bifurcated approach.

  The first approach with exhaustive analysis of sequence conservation across the Fab variable and constant interfaces was performed via alignment of known antibody sequences and structures. The alignment of the constant and variable domain sequences of various antibody subgroups is shown in FIG. FIG. 1A shows an alignment of a representative human VH germline subgroup. FIG. 1B shows an alignment of a representative human kappa VL germline subgroup. FIG. 1C shows an alignment of a representative human lambda VL germline subgroup. FIG. 1D shows an alignment of human CH1 allelic sequences. FIG. 1E shows an alignment of human kappa and lambda allele sequences. The second approach involved analysis of the D3H44 crystal structure interface using a number of molecular modeling tools (eg, Residue Contacts ™), as shown in FIG. These analyzes led to the identification of a list of hot spot locations for genetic engineering of preferential HL pairs. The hot spot positions determined in these analyzes are listed in Table 2. These positions and amino acids are primarily framework residues (except that some are located in the CDR3 loop) and most are conserved in the lambda light chain. The amino acids of the parent D3H44 sequence with Kabat numbering are presented in Tables 3a-3b.

  Next, possible mutations in the hot spot position and the position adjacent to the target hot spot in the three-dimensional crystal structure were simulated and identified via computer mutagenesis and stuffing / modeling with Zymepack ™ . Zymepack (TM), given an input structure and a set of mutations, changes the type of residues in the input structure according to the supplied mutation and generates a new structure that approximates the physical structure of the mutant protein It is a software suite. In addition, Zymepack evaluates the properties of muteins by calculating various quantitative metrics. These metrics include steric and electrostatic complementarity measurements, which can correlate with the stability, binding affinity or heterodimer specificity of the mutein.

  FIG. 3 represents a subset of hotspot positions at the variable domain heavy and light chain interfaces, and how mutations are introduced at these interface positions to drive selective pairing of absolute chains; At the same time, it has been demonstrated whether to reverse the formation of inaccurate strand pairing. Stereocomplementarity was modeled using computational methods including Zymepack ™ and also calculated based on energy factors such as van der Waals packing, cavitation effects and close contact with hydrophobic groups . Similarly, electrostatic interaction energy was modeled and evaluated based on the Coulomb interaction of charge, hydrogen bonding and desolvation effects. Simulate both preferred heavy and light chain pair models such as H1: L1 (or H2: L2) and inaccurate pair models such as H1: L2 (and H2: L1) obtained by introduction of the mutation of interest. Relative steric and electrostatic scores were calculated. This means that a particular mutation set has an energy superior to a preferred (absolute) heavy chain-light chain pair compared to an inaccurate (non-absolute) pair, ie greater steric and / or electrostatic complementarity. Made it possible to decide whether to bring sex. The calculated steric and electrostatic energy is a component of the free energy associated with light and heavy chain pairings. Thus, greater steric and electrostatic complementarity indicates greater free energy associated with absolute pair pairing as compared to non-absolute pair pairing. Greater steric and electrostatic complementarity results in a preferential (selective) pairing of absolute heavy and light chains compared to non-absolute pairs.

Example 2: Design Selection and Description Using the techniques described in Example 1, heavy-light chain heterodimer pairs (ie, H1-L1 and H2-) exhibiting selective or preferential pairings. L2) was designed. Heterodimers are designed in pairs, referred to as “designs” or “design sets”, and include a set of substitutions in the H1, L1, H2, and L2 chains that promote preferential pairing (Table 5). The design set was first tested as an “LCCA design” (Table 4) in which one heavy chain is co-expressed with two light chains to assess relative pairing. Amino acid substitutions are identified by referring to Tables 3a, 3b using the Kabat numbering system.

  Using the design library described in Table 30 of International Patent Application PCT / CA2013 / 050914 as a starting point, the LCCA designs shown in Table 4 and some of the design sets shown in Table 5 Identified. Some of the designs in Tables 4 and 5 are new independent designs. The core design is shown in Table 6 with the associated unique identifier. Most designs are only in the constant region, but some designs also incorporate modifications in the variable region. These designs were proposed to further induce pairing specificity and at the same time have a favorable transition to other antibody systems.

  In the derived design, the library of designs described in Table 30 of International Patent Application PCT / CA2013 / 050914 was used as a starting point, the designs were clustered by structural similarity, and by differential scanning calorimetry (DSC) Ranking was based on the strength of the paired specificity measured, the effect and stability of antigen binding. The designs were then combined (eg, see Table 7) and / or optimized (eg, see Tables 8 and 9) to produce a derived design. In combination, at least one design showed a high pairing specificity and the other design showed a series of favorable pairing specificities. All designs selected for combination and / or optimization showed minimal / no effect on antigen binding and showed minimal / no effect on melting temperature (Tm) Not shown.

  Independent designs are categorized as independent (classified as independent in the design type column of Table 5) or in combination with derived designs (categorized as independent / combination in the design type column of Table 5). (See also Table 10).

  The design was packed into a molecular model of D3H44 and metrics were calculated (as described in Example 1). The top design was then selected based on risk (possible effects on stability and immunogenicity) and impact (considering the proposed strength to induce pairing specificity). These high-level designs are shown in Table 5.

Example 3: Preparation of Fab constructs encoding D3H44 IgG heavy chain and D3H44 IgG light chain Wild-type Fab heavy and light chains of anti-tissue factor antibody D3H44 were prepared as follows. D3H44 Fab light chain (AJ308087.1) and heavy chain (AJ3088068.1) sequences were taken from GenBank (Table 3c), genes were synthesized, and codons were optimized for mammalian expression. The light chain vector insert consisting of 5'-EcoRI cleavage site-HLA-A signal peptide-HA or FLAG tag-light chain Ig clone-'TGA stop'-BamH1 cleavage site-3 'was ligated into the pTT5 vector (Durocher). Y et al., Nucl. Acids Res. 2002; 30, No. 2e9). The resulting vector + insert was sequenced to confirm the correct reading frame and sequence of the coding DNA. Similarly, 5'-EcoR1 cleavage site-HLA-A signal peptide - consisting of (termination, see Table 3a in _T238) -ABD 2 -His ₆ tag -TGA stop -BamH1 cleavage site 3 'heavy chain clones The heavy chain vector insert was ligated into the pTT5 vector (ABD, albumin binding domain). Also, the resulting vector + insert was sequenced to confirm the correct reading frame and sequence of the coding DNA. Various Fab D3H44 constructs containing amino acid substitutions for the design set were generated by gene synthesis or by site-directed mutagenesis (Braman J, Pawworth C & Greener A., Methods Mol. Biol. (1996) 57. : 31-44).

To drive the evaluation of preferential pairings by competition assay SPR sorting, the heavy and light chains were tagged at the C-terminus and N-terminus, respectively. ABD ₂ -His ₆ duplex tag, specifically allowing the H-L complex is captured in the anti-His tag SPR chip surface, whereas, FLAG and HA light chain tag relative L1 and L2 populations Quantification of

Example 4: Evaluation of preferential pairings of Fab heterodimers comprising either constant domain modifications or a combination of constant and variable domain modifications in D3H44 IgG light and / or heavy chains. Amino acids of the LCCA design set of Table 12 Constructs encoding D3H44 IgG heavy and light chains in a Fab format with modifications were prepared as described in Example 3. The ability of the construct to preferentially pair and form the desired H1-L1 heterodimer in the context of the LCCA design set (H1, L1, L2) was determined by use of a light chain competition assay (LCCA).

  LCCA quantifies the relative pairing of one heavy chain and at least two unique light chains and can be summarized as follows. One D3H44 heavy chain Fab construct is co-expressed with two unique D3H44 light chain Fab constructs and the relative light chain pairing specificity (eg, H1-L1: H1-L2) determined by competition assay-SPR sorting This was done twice. Reduce the amount of L1 (designed to preferentially pair with the H chain) compared to L2 (eg, L1: L2 = 1: 3 based on weight), while the heavy chain has a limited amount By maintaining (ie, H1: L1 + L2 at 1: 3), the LCCA selection ratio was biased to identify strong drivers. The amount of each heterodimer formed (ie, H1-L1 and H1-L2) is determined by binding heavy chains to the SPR chip via His tag pulldown, followed by antibodies specific for these tags. Determined by detection of the amount of each light chain tag (HA or FLAG) used. Subsequently, selected heterodimer hits were verified through validation of the light chain competition assay, which indicated that the L1: L2 DNA ratio was 1: 3 and 1: 9 upon transfection. While the heavy chain was retained in a limited amount. Note also that the light chain tag (HA or FLAG) does not affect LCCA pairing in the D3H44 system (see Example 10 of International Patent Application PCT / CA2013 / 050914). A schematic diagram of the design of this assay is shown in FIG. FIG. 5 shows how heavy and light chains are tagged and how preferential pairing is evaluated. Details of the LCCA experiments are provided below.

Transfection method CHO-3E7 cells were transfected with an LCCA design comprising one heavy chain and two light chain constructs prepared as described in Example 3 as follows. CHO-3E7 cells were cultured in FreeStyle ™ F17 medium (Invitrogen catalog number A-1383501) supplemented with 4 mM glutamine and 0.1% Koliphor P188 (Sigma # K4894) at 1.7-2 × 10 ⁶ cells / cell. The cells were cultured at 37 ° C. with a density of ml. A total volume of 2 ml was transfected with a total of 2 μg of DNA at a DNA: PEI ratio of 1: 2.5 using PEI-pro (Polyplus transfection number 115-375). Twenty-four hours after adding the DNA-PEI mixture, the cells were transferred to 32 ° C. Supernatants were tested for expression on day 7 by non-reducing SDS-PAGE analysis, followed by visualization of bands by Coomassie blue staining. The H: L ratio is as shown in Table 11.

Competition Assay SPR Method The extent of preferential D3H44 light chain pairing to D3H44 heavy chain in the LCCA design was assessed by using SPR-based readings of unique epitope tags located at the N-terminus of each light chain.

  Supply of surface plasmon resonance (SPR). GLC sensor chip, Biorad ProteOn amine coupling kit (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sNHS) and ethanolamine), and 10 mM sodium acetate buffer The solution was obtained from Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON). 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) buffer, ethylenediaminetetraacetic acid (EDTA) and NaCl were purchased from Sigma-Aldrich (Oakville, ON). The 10% Tween 20 solution was purchased from Teknova (Hollister, CA).

  SPR biosensor assay. All surface plasmon resonance assays were performed using BioRad ProteOn XPR36 instrument (Bio-Rad Laboratories (Canada) Ltd.) (Mississauga, ON) with PBST running buffer (running buffer) (PBS, Teknova Inc, 0.05% Tween 20). ) At a temperature of 25 ° C. An anti-pentaHis capture surface was generated using a GLM sensor chip activated with a 1: 5 dilution of a standard BioRad sNHS / EDC solution injected at 100 μL / min in the analyte (horizontal) direction for 140 seconds. Immediately after activation, a 25 μg / mL solution of anti-pentaHis antibody (Qiagen Inc.) in 10 mM NaOAc, pH 4.5 was added at approximately 3000 resonance units (flow) in the analyte (horizontal) direction at a flow rate of 25 μL / min. RU) was injected until immobilized. The remaining active groups are stopped by injecting 1M ethanolamine for 140 seconds at a flow rate of 100 μL / min in the direction of the analyte, which creates a pseudo-activated interspot for the blank reference I also made sure.

  Selection of heterodimers for binding to anti-FLAG (Sigma Inc.) and anti-HA (Roche Inc.) monoclonal antibodies was performed in two steps. Indirect capture of the heterodimer in the ligand direction to the anti-penta His surface, followed by injection of anti-FLAG and anti-HA in the analyte direction. Initially, a single injection of 100 μL / min 30 seconds PBST in the ligand direction was used for baseline stabilization. For each heterodimer capture, the unpurified heterodimer in cell culture medium was diluted to 4% with PBST. 1-5 heterodimers or controls (ie controls containing 100% HA light chain or 100% FLAG light chain) are injected simultaneously into individual ligand channels at a flow rate of 25 μL / min for 240 seconds and saturated Approximately 300-400 RU of heterodimer capture was brought to the anti-penta His surface. The first ligand channel was left empty and used as a blank control as needed. Immediately following this heterodimer capture step, injection of two buffers in the direction of the analyte followed by stabilization of the baseline, followed by 5 nM anti-FLAG and 5 nM anti-HA, respectively 180 Injection for 120 seconds at 50 μL / min, with a second dissociation step, was repeated twice, resulting in a set of binding sensorgrams using a buffer reference for each of the captured heterodimers. Tissue factor (TF) antigen to which the heterodimer binds was also injected as an activity control into the last remaining analyte channel. The heterodimer was regenerated for 18 seconds at 100 μL / min with an 18 second pulse of 0.85% phosphoric acid to prepare an anti-pentaHis surface for the next injection cycle. Sensorgrams were aligned and double-referenced by using buffer blank injection and interspots, and the resulting sensorgrams were analyzed by using ProteOn Manager software v3.0.

Results The results of LCCA are shown in Tables 12, 13a and 14a. In Tables 13 and 14, “unique identifier” indicates that the unique identifier of the two configuration LCCAs is in any orientation ((H1L1L2 set number−H2L2L1 set number) or (H2L2L1 set number−H1L1L2 set number)) Note that this may not exactly match Table 5. Evaluation of the preferential pairing of each LCCA design is shown in the three columns after Table 12. The same data is also included in the context of design pairs in columns 5, 6 and 8 or 10, 11 and 13 of tables 13a and 14a. Each unique set of H1, L1 and L2 mutations (LCCA design) was assigned a unique number or “set number” (eg, 9567 or 9087). When data is represented in the H1L1H2L2 format (Fab vs format or design set), such a design set is thus indicated by a “unique identifier” composed of the set number of two components LCCA (eg 9567-9087). It is. Note that most LCCA experiments were performed on constructs containing interchain Fab disulfide bonds located in the constant domain (H / C233-L / C214, Kabat numbering). In Tables 13 (a and b) and 14 (a and b), two complementary LCCA sets (H1, L1, L2 and H2, L2, L1) are used to highlight specific designs that were successful with respect to preferential matching. ) Is expressed in the Fab pair format. The presence of tags (L chain: HA and FLAG, and H chain: ABD ₂ -His ₆ ) did not affect about 50%: 50% predicted neutral pairing in D3H44 WT.

  In this table, the reported LCCA data is the median of ratio formats (H1-L1: H1-L2 and H2-L2: H2-L1) normalized to L1: L2 DNA ratio = 1: 1. In addition, LCCA data was normalized to 100% because the total amount of L1 and L2 was observed to be significantly different from 100% in some variants. This difference in the total light chain ratio may be due in part to the occurrence of non-specific binding variations upon initial heterodimer capture on the SPR chip. Since LCCA experiments were performed with two different L1: L2 DNA ratios (L1: L2 of 1: 3 and 1: 9, respectively), both LCCA normalization ratios are presented in the table. LCCA data was obtained when the experimental data obtained did not meet the criteria for inclusion (eg, SPR chip Fab capture was less than 100, or the total LCCA of L1 and L2 was outside the range of 60-140. Therefore, some LCCA experiments were not reported.

  Table 12 covers all of the LCCA designs (530) for which data was obtained. Of the 530 LCCA designs, 490 (92.5%) of these LCCAs are at least 60% accurate pairings (L1: L2 DNA ratio = 1: 3 and 1: 9) L1: L2 DNA ratio = 1: 1 normalized). The remaining LCCA designs are mainly neutral LCCA designs (32/530 or 6.0%), as well as a small percentage of LCCA designs that produced inconsistent results (8/530 or 1.%). 5%). The designs shown in Table 12 are primarily electrostatic (based on specific drivers utilizing hydrogen bonding or charge-charge interactions), and some designs are steric complementarity and Also includes interchain covalent disulfide bonds. Some designs also included mutations for the formation of new disulfide bonds (formed by H / C233-L / C214) in the absence of natural interchain disulfide bonds.

  Tables 13 (a and b) and 14 (a and b) cover 447 designs with LCCA data present in both heterodimers of the design set. Tables 13a and 14a show that the computer approach described in Example 1 shows that H1-L1 preferential pairing over H1-L2 and H2-L2 over H2-L1 over various design sets and variants. Demonstrates the achievement of preferential matching.

  Table 13 (a and b) covers designs with average LCCA performance with Fab heterodimer pairing: mispairing of at least 86:14 (L1: L2 ratio normalized median is H1 -L1: H1: L2 and H2-L2: 1: 1 for H2-L1), while Table 14 (a and b) shows Fab heterodimer pairing: mispairing less than 86:14 The design with average LCCA performance is covered. The performance of each LCCA was normalized to 100%, as well as the L1: L2 DNA ratio was normalized to 1: 1 (described above this example), and both scalar values ((ln (r1 / f1) Or ln (r2 / f2)), where r1 and r2 correspond to the median of H1L1: H1L2 and H2L2: H2L1, respectively, in experimental ratio, and f1 and f2 correspond to the corresponding experimental ratio ), Described as the ratio of paired and mispaired Fab heterodimers. Each design also has an associated average LCCA performance scalar value (0.5 (ln (r1 / f1) + ln (r2 / f2))), which is also normalized to 100%, as well as L1: L2 DNA The ratio is also normalized to 1: 1 (described above this example). In addition, the scalar range of each LCCA of the design (LCCA1 and LCCA2 corresponding to H1L1: H1L2 and H2L2: H2L1) is shown. Of the 447 Mab designs, 354 (79.2%) show an average LCCA performance of at least 86:14 (Tables 13a and b). The designs in Table 13 (a and b) were further characterized into 13 clusters based on design similarity. The designs within each cluster were arranged by highest to lowest average LCCA performance scalar value.

In addition, the LCCA data in Table 13a is also displayed graphically in FIG. FIG. 7 shows a box plot of average LCCA performance values with Fab heterodimer pairing: mispairing at least 86:14 in each cluster. The lower part of the box chart shows the first quartile (Q1), which is the minimum and median so that values below the first quartile represent at least 25% of the data. Intermediate average LCCA performance value. The horizontal bar in the box plot indicates the second quartile, which is the median average LCCA performance value for the cluster. The top of the box chart shows the third quartile (Q3), which is the maximum and median so that values above the third quartile represent up to 25% of the data Is an intermediate average LCCA performance value. The interquartile region is the difference between Q3 and Q1. Whiskers that extend vertically in both directions indicate a data range of values that are both within Q1- (1.5 ^* IQR) or Q3 + (1.5 ^* IQR). The horizontal bar that caps the shoreline indicates the maximum and longest values in the range. Data and Higesen exists outside the Hakohigezu plot identified as outliers, central outliers is indicated by dotted lines (different Q1 or Q3 and ^{1.5 * IQR~3} * IQR), extreme outliers , + Sign (different from Q1 or Q3 over 3 ^* IQR).

Example 5: Scale-up of biophysical characterization To test precisely matched heterodimers as indicated by a unique identifier set (Table 5) for thermal stability and antigen binding, the following: To a large scale (typically 20 ml) and purified. Heterodimeric heavy and light chains were expressed in 20 ml cultures of CHO-3E7 cells. CHO-3E7 cells are 1.7-2 × 10 ⁶ cells in FreeStyle ™ F17 medium (Invitrogen catalog number A-1383501) supplemented with 4 mM glutamine and 0.1% Koliphor 188 (Sigma # K4894). Cultured at 37 ° C. with a density of / ml. The total volume of 20 ml was transfected with a total of 20 μg of DNA at a DNA: PEI ratio = 1: 2.5 by using PEI-pro (Polyplus catalog number 115-375). Twenty-four hours after adding the DNA-PEI mixture, the cells were transferred to 32 ° C.

  Cells were centrifuged 7 days after transfection and the heterodimer was purified from the supernatant by high-throughput nickel affinity chromatography purification as follows. The supernatant is diluted to 20-25% cell culture supernatant with equilibration buffer (calcium, magnesium and phenollet (Dulbecco-phosphate buffered saline (DPBS) without HyClone ™ number SH30028.02), then And incubated for 12 hours with mixing in HisPur® Ni-NTA resin (Thermo Scientific number PI-88222), previously equilibrated in equilibration buffer.The resin is then collected by centrifugation, 96 Transfer to a well frit plate, wash 3 times with equilibration buffer and elute using HIS-Select® elution buffer (Sigma-Aldrich number H5413).

  After purification, heterodimeric expression was assessed by a non-reducing High Throughput Protein Express assay using Caliper LabChip GXII (Perkin Elmer # 760499). The procedure was performed according to HT Protein Express LabChip User Guide version 2 LabChip GXII User Manual with the following changes. Heterodimer samples were transferred to separate wells of a 96 well plate (BioRad # HSP9601) in either 2 μl or 5 μl (concentration range 5-2000 ng / μl) to 7 μl of HT Protein Express Sample Buffer (Perkin Elmer # 760328). ) The heterodimer sample was then denatured at 70 ° C. for 15 minutes. The LabChip instrument was operated by using HT Protein Express LabChip (Perkin Elmer # 760499) and Ab-200 assay settings. After use, the chip was cleaned with MilliQ water and stored at 4 ° C.

Example 6: Measurement of Thermal Stability of Fab Heterodimers by DSF To assess thermal stability, differential scanning fluorescence measurement (DSF) was used as a high-throughput method, using wild-type unmodified heavy chain- All correctly paired heterodimers were selected as compared to the light chain pair. The heterodimer was prepared as described in Example 5.

Measurement of thermal stability The thermal stability of all heterodimer pairs was measured using DSF as follows. Each heterodimer was purified as described in Example 5 and diluted to 0.5 mg / mL with DPBS (HyClone catalog number SH30028.02). In most samples, a working stock of Sypro Orange gel stain (Life Technologies catalog number S-6650) was prepared by diluting 4 μL of Sypro Orange gel stain in 2 ml of DPBS. DSF samples were prepared by adding 14 μL of 0.5 mg / mL protein to 60 μL of diluted Sypro Orange gel staining working solution. However, for proteins below 0.5 mg / mL, each DSF sample was prepared by adding 14 μL of undiluted protein to 60 μl of Sypro Orange dye working solution (diluted 1: 1500 with DPBS). DSF analysis was then performed in duplicate in 20 μl aliquots by use of a Rotor-Gene 6000 qPCR instrument (QiaGen Inc). Each sample was scanned from 30 ° C. to 94 ° C. using a 1 ° C. interval with 10 seconds equilibration and 30 seconds waiting time at the start of each step. A 470 nM excitation filter and a 610 nM emission filter with a gain of 9 were used. Data were analyzed by Rotor-Gene 6000 software using the maximum value from the first derivative of the Tm denaturation curve. The remaining DSF samples were similarly prepared and analyzed by the following protocol modifications that did not change the measured Tm values. 1) Working solution was prepared by diluting 1 μL of Sypro Orange gel stain in 2 ml DPBS. 2) A 30 μl aliquot was analyzed. 3) A gain of 10 was used.

  The DSF results are shown in Tables 12, 13b and 14b. The thermal stability of the H1: L1 Fab in the context of LCCA design (DSF value and DSF value compared to wild type) is shown in columns 3 and 4 of Table 12. The same DSF value is also included in the columns 7 and 8 of Tables 13b and 14b in the context of the design pair. The reported Tm value is median for each Fab heterodimer where the iterations are performed. Comparison of the Tm value of the Fab heterodimer with the Tm value of the wild-type Fab heterodimer (wild-type Fab construct containing the HA tag and having a median Tm of 81.0 ° C.) was compared to the column of H1L1_dTm_dsf. To report to. In some Fab heterodimers lacking the natural interchain disulfide bond (between the H chain C233 and L chain C214), the H1L1_dTm_dsf value indicates the corresponding wild type Fab lacking the natural interchain disulfide. Note that it was not determined because it was not evaluated. Also, some Fab heterodimers have a corresponding experimental quality (eg, low yield, low intensity, partially masked peak and variation between Fab heterodimer repeats above 1 ° C. Note that Tm values were not reported (17/230 or 7.4% Feb heterodimer) due to In some of these Fab heterodimers, an estimated Tm value corresponding to the Tm value of a similar Fab heterodimer that differs only in the presence / absence or predisposition of a bound light chain tag (HA or FLAG) is Reported on. For the estimated Tm value, the corresponding wild-type Tm value (81.2 ° C.) is the median value obtained from all wild-type Fab heterodimeric constructs (ie, Fab constructs containing HA or FLAG tags). . The HA or FLAG tag does not significantly affect the Tm value of the wild type Fab heterodimer. Overall, Fab heterodimers showed similar Tm values compared to WT. Of Fab heterodimers that contain natural interchain disulfides and for which DSF data are also available, 93% (195/209) of Fab heterodimers lost less than 3 ° C compared to WT showed that. Furthermore, the most affected Fab heterodimer showed a loss of 6.5 ° C. compared to WT. Table 12 covers the LCCA designs in order of decreasing Tm rank.

  In addition, 13 amino acid substitutions have been identified that generally improve the stability of Fab heterodimers (see Table 34). Stabilizing mutations were identified after comparison of Fab heterodimers containing stabilizing mutations with similar Fab heterodimers that differed in the absence of stabilizing mutations. Heavy chain stabilizing mutations include A125R, H172R, L143F, Q179D, Q179E, Q39R, S188L and V190F. Light chain stabilizing mutations include Q124E, Q124R, Q160F, S176L and T180E. Overall, the stabilizing mutation increased stability by 0.4 ° C to 2.1 ° C. Heavy chain stabilizing mutations A125R, H172R, L143F, Q179D, Q179E, Q39R, S188L and V190F have a stability of 0.4 ° C. to 0.6 ° C., 0.4 ° C. to 2.1 ° C., 0. Increased 4 ° C, 0.5 ° C to 0.6 ° C, 0.5 ° C to 0.8 ° C, 1.1 ° C to 1.6 ° C, 0.4 ° C to 1.2 ° C and 1 ° C. Light chain stabilizing mutations Q124E, Q124R, Q160F, S176L and T180E have a stability of 0.4 ° C to 0.5 ° C, 0.8 ° C to 0.9 ° C, 0.6 ° C and 0.4 ° C, respectively. Increased by -1.0 ° C and 0.5 ° C.

Example 7: Measurement of Fab Heterodimer Antigen Affinity Does Fab Heterodimer have any effect on the ability to bind to tissue factor and the ability of amino acid substitutions to bind to the heterodimer antigen? Evaluated to determine. The affinity of each Fab heterodimer to tissue factor was determined by SPR as follows.

  SPR supply. GLC sensor chip, Biorad ProteOn amine coupling kit (EDC, sNHS and ethanolamine), and 10 mM sodium acetate buffer were purchased from Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON). PBS running buffer with 0.05% Tween 20 (PBST) is available from Taknoca Inc. (Hollister, CA).

  Batch of Fab heterodimers. Purified Fab heterodimers were tested in three batches A, B and C. Batches A and B are stored for approximately 1 month at 4 ° C. prior to performing the SPR assay, while the batch C Fab heterodimer is approximately 2 ° C. at 4 ° C. prior to performing the SPR assay. Saved for months. Batch C Fab heterodimers are marked with a “+” next to the corresponding KD value in Table 12.

  All surface plasmon resonance assays were performed using a BioRad ProteOn XPR36 instrument (Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON)) with PBST running buffer (running buffer). The anti-pentaHis capture surface was generated using a GLC sensor chip activated with a 1.5 dilution of a standard BioRad sNHS / EDC solution injected at 100 μL / min in the analyte (horizontal) direction for 140 seconds. Immediately after activation, a 25 μg / mL solution of anti-pentaHis antibody (Qiagen Inc.) in 10 mM NaOAc, pH 4.5 was added at approximately 3000 resonance units (flow) in the analyte (horizontal) direction at a flow rate of 25 μL / min. RU) was injected until immobilized. The remaining active groups are stopped by injecting 1M ethanolamine for 140 seconds at a flow rate of 100 μL / min in the direction of the analyte, which creates a pseudo-activated interspot for the blank reference I also made sure.

  Selection of Fab heterodimers that bind to the TF antigen was performed in two steps. Simultaneous capture of 5 purified antigen concentrates and 1 buffer blank for indirect capture of Fab heterodimer on anti-pentaHis antibody surface in ligand direction, followed by double reference in analyte direction It is an injection. Initially the baseline was stabilized by a 30 second injection at 100 uL / min in the direction of the ligand in one buffer. 1 to 5 variants or controls at a concentration of 3.4 μg / ml in PBST were co-injected into individual ligand channels for 240 seconds at a flow rate of 25 μL / min. This resulted in approximately 1000 RU average capture on the anti-penta His surface in batches A and B, and in batch C approximately 600 RU average capture on the anti-penta His surface. The first ligand channel was left empty and used as a blank control as needed. Immediately following this capture step, two buffer injections at 100 μL / min in the analyte direction for 30 seconds each followed to stabilize the baseline and then 60 nM, 20 nM, 6.7 nM, 2.2 nM and 0.74 nM antigen (TF) was injected simultaneously at 50 μL / min for 120 seconds with a buffer blank with a 600 second dissociation step. The capture antibody surface was regenerated for 18 seconds at 100 μL / min with an 18 second pulse of 0.85% phosphoric acid twice and prepared for the next injection cycle. Sensorgrams were aligned and double-referenced by using buffer blank injection and interspots, and the resulting sensorgrams were analyzed by using ProteOn Manager software v3.1. A double reference sensorgram was fitted to the 1: 1 binding model. The Rmax value of each antigen was normalized to the antibody capture level of each variant and compared to a 100% control.

  The antigen affinity (KD) values of Fab heterodimer samples are reported in Tables 12, 13b and 14b. The KD values of H1: L1 Fab in the context of LCCA design (range of KD and KD values and KD median value compared to wild type) are shown in columns 5, 6 and 7 of Table 12, respectively. The same KD values are given in columns 13 (H1-L1 Fab heterodimer KD), 4 (KD change of H1-L1 Fab heterodimer compared to wild type), 5 (H2-L2) in Tables 13b and 14b. KD of Fab heterodimer), as well as 6 (K2 change of H2-L2 Fab heterodimer compared to wild type) in the context of the design pair. KD values were determined only in Fab heterodimer samples that showed at least 100 RU Fab heterodimer capture. Reference wild type KD (0.157 nM) reflects the median of wild type Fab heterodimers where the light chain contains a FLAG tag. Wild-type Fab heterodimers (containing either FLAG or HA tags) showed similar KD values, with a 2.6-fold difference observed between the maximum and minimum values. In Tables 12, 13b and 14b, the difference in KD for wild-type antigen binding affinity is shown using the calculation-(logarithmic (KD) design-logarithmic (KD) wt), with positive values for antigen Compared to the wild-type binding affinity, the Fab heterodimer shows a reduced KD value, while the negative value shows an increased KD value. Note that some Fab heterodimers lack measured KD values. In some of these cases, Fab heterodimers were evaluated, but SPR experiments showed low Fab heterodimer capture (ie, less than 100 RU) and therefore accurate determination of KD values is not possible It was. For Fab heterodimers that show similarity to other Fab heterodimers (ie, differ only in the presence / absence or predisposition of bound light chain tag (HA or FLAG)), similar Fab heterodimers Instead, an estimated KD value corresponding to the KD value is provided (shown in Tables 12, 13b and 14b). The corresponding estimated wild type KD value (0.15 nM) was the median value obtained from all wild type Fab heterodimer constructs (ie, Fab constructs containing HA or FLAG tags). Overall, the results show that correctly paired heterodimers (from a design point of view) show a wild-type-like binding affinity for antigen (within about 2.3 times the reference wild-type affinity). It is shown.

Example 8 FIG. Ultra High Performance Liquid Chromatography Size Exclusion Chromatography (UPLC-SEC) Profile of Wild Type Tagged D3H44 Heterodimer and Preferentially Paired Heterodimer on C-terminal ABD2-His ₆ Tag and Light Chain Wild-type D3H44 heterodimers (one heavy chain and one light chain) with an N-terminal tag (FLAG in one construct and HA in another construct) are described in known methods of the art and in Example 5. Expressed and purified by methods similar to those described. Preferential or precisely paired heterodimers were individually scaled up and purified by His tag affinity purification as described in Example 5.

  Using a Waters BEH200 SEC column (2.5 mL, 4.6 × 150 mm, stainless steel, 1.7 μm particles) set at 30 ° C. and attached to a Waters Acquity UPLC system with a PDA detector, the UPLC-SEC was Carried out. The run time consisted of 7 minutes, the total volume per injection was 2.8 mL, and the running buffer Hyclone DPBS / modified-calcium-magnesium (part number SH30028.02) was used at 0.4 ml / min. The elution was monitored by UV absorption ranging from 200 to 400 nm and the chromatogram was extracted at 280 nm. Peak integration was performed using Empower3 software.

  FIG. 6 shows a representative WT Fab heterodimer pair (contains a FLAg tag in the light chain), and a representative of the designed Fab heterodimer pair (LCCA designed 9735, 9737 and 9740 H1L1 Fab components). This is a UPLC-SEC profile. In general, the designed Fab heterodimer pairs showed a similar UPLC-SEC profile compared to WT.

Example 9: Evaluation of preferential pairing of heterodimers in a co-expression set comprising constant domain or constant and variable domain modifications in a bispecific antibody format. Was also evaluated to determine if specific pairings were possible. In this example, in order to promote the heterodimerization of the unique heavy chain, the full-length heavy chain Fc region of each heterodimer, one modified with modified T350V, L351Y, F405A and Y407V. And the other heavy chain was asymmetrically modified to include modifications T350V, T366L, K392L and T394W (EU numbering).

Preparation of the construct:
Heterodimeric designs were tested in the context of the following bispecific antibodies: a) D3H44 / trastuzumab, b) D3H44 / cetuximab, and c) trastuzumab / cetuximab. Note that D3H44 is a human antibody, trastuzumab is a humanized antibody, and cetuximab is a chimeric antibody containing human IgG1 and mouse Fv regions. Constructs encoding D3H44, trastuzumab and cetuximab IgG heavy and light chains, including amino acid modifications by design, were prepared as follows. The base DNA sequences of the heavy and light chains of D3H44, trastuzumab and cetuximab are shown in Table 3C. D3H44, trastuzumab and cetuximab light chain sequences were prepared as described in Example 3 except that some sequences lacked tags but others contained FLAG or HA tags. . The D3H44, trastuzumab and cetuximab heavy chain sequences are created and modified by adding the IgG1 ^* 0.1 DNA sequence in which the full-length heavy chain encodes the hinge CH2-CH3 domain to the C1 of the CH1 domain of the Fab heavy chain. Prepared as described in Example 3, except that dimerization was promoted at the ends. The basic C-terminal heavy chain lysine residue was removed to prevent LC-MS signal heterogeneity due to C-terminal lysine clipping (Lawrence W. Dick Jr. et al., Biotechnol. Bioeng. (2008) 100: 1132-43).

Assay format (SMCA)
The ability of heterodimer co-expression set designs to preferentially pair to form bispecific antibodies was evaluated as described below. The assay co-expressed four chains (H1 and L1 chains from one antibody, H2 and L2 chains from the other antibody) and mass spectrometry (LC-MS) for the presence of correctly formed bispecific antibodies. ) Based on detection. FIG. 8 shows four starting polypeptide chains and the potential products resulting from the co-expression of these starting polypeptides in the absence of preferential pairing of the heavy and light chains of the heterodimer pair. The schematic shown is provided. The two full-length heavy chain constructs are co-expressed with the two unique light chain constructs to generate 10 possible antibody species: H1-L1: H1-L1, H1-L2: H1-L2, H1-L1: H1-L2, H2-L1: H2-L1, H2-L2: H2-L2, H2-L1: H2-L2, H1-L1: H2-L1, H1-L2: H2-L2, H1-L2: H2- L1 and H1-L1: yielded H2-L2. The H1-L1: H2-L2 species are precisely paired bispecific antibodies (see FIG. 8). The relative pairing specificity relative to others for the amount of H1-L1: H2-L2 of the preferred species was determined using LC-MS after pA purification and deglycosylation. If possible, the strands were left untagged, provided that all Mab and half-Ab species differed from each other by at least 50 Da. When the mass difference deviated from this possibility, an N-terminal tag (HA or FLAG) was added to the light chain to provide a sufficient mass difference between the species.

  This assay with bispecific antibody expression and sorting steps is called SMCA.

Mass Spectrometry The extent of preferential D3H44 light chain pairing to D3H44 heavy chain in the co-expression set was assessed using mass spectrometry after protein A purification and non-denaturing deglycosylation. Since D3H44 / trastuzumab heterodimer contains only Fc N-linked glycans, this system was treated with only one enzyme, N-glycosidase F (PNGase F). The purified sample was deglycosylated with PNGase F as follows. 0.2U PNGase F / μg antibody in 50 mM Tris-HCl, pH 7.0 was incubated overnight at 37 ° C. to a final protein concentration of 0.5 mg / mL. In the D3H44 / cetuximab and trastuzumab / cetuximab systems, the system was treated with N-glycosidase F + multiple exoglycosidases due to the additional N-linked glycans in the Fab region of cetuximab. Typically, four enzyme mixtures were used for this purpose. N-glycosidase F, β-galactosidase (Prozyme), β-N-acetylglucosaminidase (New England Biolabs) and neuraminidase. N-glycosidase F removes Fc N-linked glycans, while exoglycosidase cleaves Fab N-linked glycans into uniform core structure M3F (GlcNAc ₂ Man ₃ Fuc ₁ ). The purified sample was deglycosylated with the four enzyme mixtures as follows. 0.2 U PNGase F / μg antibody, 0.002 U α-neuraminidase / μg antibody, 0.0001 U β-galactosidase / μg antibody and 0.2 U β-N-acetyl in 50 mM Tris-HCl pH 7.0 Glucosaminidase / μg antibody was incubated overnight at 37 ° C. to a final protein concentration of 0.5 mg / mL. However, in some cases, three enzymatic treatments (N-glycosidase F, β-galactosidase and neuraminidase) were preferred to avoid mass duplication of sample components in LC-MS analysis. In these cases, the Fab glucan was reduced to a slightly larger structure G0F (Man ₃ GlcNAc ₂ Fuc ₁ GlcNAc ₂ ). The purified sample was deglycosylated with the three enzyme mixtures using the same concentrations and conditions as described for the four sample mixtures. After deglycosylation, samples were stored at 4 ° C. prior to LC-MS analysis.

  Deglycosylated protein samples were analyzed by untreated LC-MS using an Agilent 1100 HPLC system coupled to an LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific) via an Ion Max electrospray ion source (ThermoFisher). Sample (5 μg) was injected onto a 2.1 × 30 mm Poros R2 reverse phase column (Applied Biosystems) and the following gradient conditions: 0-3 minutes with 20% solvent B, 3-6 minutes with 20-90% solvent B Using 90-20% solvent B for 6-7 minutes and 20% solvent B for 7-9 minutes. Solvent A was degassed 0.1% formic acid aqueous solution and solvent B was degassed acetonitrile. The flow rate was 3 mL / min. The flow was split after the column and 100 μL was directed to the electrospray interface. The column was heated to 82.5 ° C. and the solvent was pre-column heated to 80 ° C. to improve the protein peak shape. LTQ-Orbitrap XL was calibrated by using ThermoFisher Scientific's LTQ-Positive Ion ESI calibration solution (caffeine, MRFA and Ultramark 1621) and adjusted by use of a solution of 10 mg / mL CsI. The cone voltage (source fragmentation setting) was 40 V, the FT decomposition was 7,500, and the scan range was m / z 400-4,000. LTQ-Orbitrap XL was adjusted for optimal detection of target protein (> 50 kDa).

  Ranges containing multiple charged ions from full size antibodies (m / z 2000-3800) and half antibodies (m / z 1400 to 2000), MassLynx MaxEnt 1 module (Waters), instrument control and data analysis software. Used separately to deconvolute to a molecular weight profile. Briefly, raw protein LC-MS data is first opened with Xcalibur's spectral observation module QualBrowser (Thermo Scientific), converted using Databridge, a file conversion program provided by Water, Adapted to MassLynx. Transformed protein spectra were observed with MassLynx's Spectrum module and deconvolved with the use of MaxEnt 1. The abundance of different antibody species in each sample was determined directly from the resulting molecular weight profile.

Representative Designs in SMCA Assay A total of 25 representative designs with high average LCCA performance values were selected from clusters 1-12 for testing in the SMCA format. A representative design was selected based on a corresponding set of designs that occupy similar space, using similar drivers but also sharing similar mutations. At least one representative design was selected from each cluster. Some clusters were represented by one representative design (ie, clusters 1, 5, 7, 8, 10). The remaining clusters have more than one representative design because the clusters are large (ie, cluster 2) or composed of small clusters (ie, clusters 3, 4, 6, 9, 11, and 12). did. The designs within each cluster shared sequence similarity, but the small clusters within the cluster differed by at least one driver and the set of mutations. For clusters composed of small clusters, an additional representative design was selected from each small cluster.

  The amino acid substitutions in each cluster are covered in Tables 15-27 and the corresponding representatives in each cluster / small cluster are shown. In cluster 1, only one design (9134-9521) was chosen to represent the cluster because these designs utilized similar electrostatic drivers that occupy similar space (Table 15). In all members of this cluster, H1 was designed such that negative charge substitution (L124E and Q179E) can form a salt bridge with a positive charge substitution of L1 (either S176R and S131K or S131R). H2 was designed such that a positive charge substitution (either L124R and Q179K or S186K) can form a salt bridge with a negative charge substitution of L2 (either S176D and T178D or T178E, and / or T180E). Incompatible pairs H1L2 and H2L1 are rejected primarily by electrostatic repulsion.

  For cluster 2, two representative designs (9279-9518 and 9286-9402) were chosen to represent large clusters (see Table 16). The design within this cluster utilized a similar electrostatic driver that occupies a similar space. In all members of this cluster, H1 is a negative charge substitution (L124E and L143E or L143D) is a positive charge substitution (S176R, and (Q124K and / or T178K) or any combination of (Q124K and Q160K)) with L1 And designed to form salt bridges. H2 was designed such that positive charge substitutions (L124R and Q179K or S186K or S186R) can form salt bridges with negative charge substitutions of L2 (S176D and T178D or T178E, and / or T180E). Incompatible pairs H1L2 and H2L1 are rejected primarily by electrostatic repulsion.

  For cluster 3, five representative designs (9338-9748, 9815-9825, 6054-9327, 9066-9335, and 9121-9373) were selected to represent five small clusters (see Table 17). . All members of this cluster utilize electrostatic drivers similar to H1 (L124E), L1 (S176R), H2 (L124R) and L2 (S176D), which are preferentially paired heterodimers Allows the formation of salt bridges in the body, while incompatible pairs are rejected primarily by electrostatic repulsion. The 6054-9327 design was chosen to represent these small clusters, primarily to represent designs that utilize steady-state drivers. In addition to these electrostatic interactions, one small cluster also contained variable region stereo drivers (L45P in H1 and P44F in L1), so representatives containing variable region drivers were chosen to represent this small cluster. (9338-9748). Another small cluster also includes variable region electrostatic drivers (Q39E for H1, Q38R for H1, Q39R for H2, Q38E for L2) in both Fab heterodimers, and therefore representatives including this variable region driver are Selected to represent this small cluster (9815-9825). In addition, one small cluster is composed of one member, so one representative design (9066-9335) contains a modified disulfide between H1 F122C and L1 Q124C. The remaining small cluster, represented by 9121-9373, slightly modifies the interaction of the H1L1 constant region driver, mainly using constant region drivers with additional substitutions of H172T in H1 and S174R in L1, and at the same time The effect of H172R on HC2 was also explored.

  For cluster 4, two representative designs (9168-9342 and 9118-6098) were chosen to represent each two small clusters (see Table 18). All members of this cluster utilize electrostatic drivers similar to H1 (L124E), L1 (S176R or S176K), H2 (L124R) and L2 (S176D), which are preferentially paired heterogeneous Allows formation of salt bridges in the dimer, while incompatible pairs are rejected primarily by electrostatic repulsion. One small cluster is represented by 9118-6098 and primarily utilizes a shared electrostatic driver for preferential pairing, while the other small cluster represented by 9168-9342 is responsible for the formation of additional salt bridges. Further substitutions of H1 (K228D) and L1 (S121K) were made available.

  Cluster 5 is represented by a unique identifier 9116-9349 and is composed of only one member (see Table 19). This design includes electrostatic drivers in H1 (L124E), L1 (S176R), H2 (L124R) and L2 (S176D), and H1 (A139W), L1 (F116A_V133G_L135V), H2 (A139G_V190A) and L2 (V133G_L135W) Both three-dimensional drivers were used. As a result, in preferentially paired heterodimers, charge substitution allows the formation of salt bridges. In mismatched Fab heterodimers, formation is rejected by electrostatic repulsion and additional steric effects.

  For cluster 6, two representative designs were chosen to represent each two small clusters (see Table 20). All members of this cluster utilize electrostatic drivers similar to the constant region (Q179E for H1, S131K for L1, S186R for H2, and Q124E, Q160E and T180E for L2), which preferentially It allows the formation of salt bridges in the combined heterodimer, while incompatible pairs are rejected primarily by electrostatic repulsion. In addition, the small cluster was composed of different variable region drivers. One small cluster was represented by the unique identifier 9814-9828 and utilized electrostatic drivers (Q39E for H1, Q38R for L1, Q39R for H2, and Q38E for L2) for the variable region. The other small cluster utilized a variable region solid driver composed of L45P at H1 and P44F at L1. As a result, in this small cluster, mismatched pairs are further rejected by the introduced steric effect. This small cluster is represented by a design derived from a unique identifier 9745-9075 that differs only in the absence of Q38E in L2.

  In cluster 7, only one design (9060-9756) was chosen to represent the cluster because these designs utilized similar electrostatic and solid drivers (see Table 21). about). The shared electrostatic driver consisted of L143E and Q179E at H1, Q124R at L1, Q179K at H2, and Q124E, Q160E and T180E at L2. The shared solid driver was composed of A139W at H1, F116A_L135V at L1, and L135W at L2. As a result, in prepaired heterodimers, charge substitution in the Fab region allows the formation of salt bridges. In mismatched heterodimers, formation is rejected by electrostatic repulsion and additional steric effects.

  In cluster 8, only one design (9820-9823) has been selected to represent the cluster, since these designs utilized similar electrostatic drivers (see Table 22). . In this variable region, Q39E was used for H1, Q38R for L1, Q39R for H2, and Q38E for L2. In the constant region, L143E was used for H1, Q124R, Q160K and T178R for L1, Q179K for H2, and Q124E, Q160E and T180E for L2. In preferentially paired heterodimers, charge substitution in the Fab region allows for the formation of salt bridges, whereas in mismatched heterodimers, formation is rejected primarily by electrostatic repulsion.

  In cluster 9, two representative designs were chosen to represent each two small clusters (see Table 23). All members of this cluster utilize electrostatic drivers similar to the constant region (L143E for H1, Q124R for L1, Q179K for H2, and Q124E, Q160E and T180E for L2), which preferentially It allows the formation of salt bridges in the combined heterodimer, while incompatible pairs are rejected primarily by electrostatic repulsion. In addition, small clusters depend on the presence / absence of variable region drivers (L45P in H1 and P44F in L1). As a result, in small clusters containing variable region drivers, incompatible pairings are further rejected by the introduced steric effect. A representative design of this small cluster containing variable region drivers was derived from the unique identifiers 9751-9065 that differ only in the absence of Q38E in L2. A representative design for each small cluster with variable region substitution is 9611-9077.

  In cluster 10, only one design (9561-9095) has been chosen to represent the cluster, because these designs utilized similar electrostatic and stereo drivers that occupy similar space. (See Table 24). The shared electrostatic driver consisted of L143E and Q179E at H1, and was similarly located at Q124R, Q124K or S131K at L1, Q179K at H2, and Q124E and T180E at L2. The shared three-dimensional driver was composed of L124W at H1, V133A at L1, and V133W at L2. As a result, in prepaired heterodimers, charge substitution in the Fab region allows the formation of salt bridges. In mismatched heterodimers, formation is rejected by electrostatic repulsion and additional steric effects.

  For cluster 11, three representative designs (9049-9759, 9682-9740, and 9667-9830) were each selected to represent three small clusters (see Table 25). All members of this cluster utilized electrostatic substitution to guide preferential pairing of heterodimers. As a result, in prepaired heterodimers, charge substitution in the Fab region allows the formation of salt bridges. In mismatched heterodimers, formation is rejected primarily by electrostatic repulsion. In small clusters represented by the unique identifiers 9667-9830, covalent substitutions are negative charge substitutions for H1 (L143E or L143D and Q179E or Q179D), positive charge substitutions for L1 (T178R or T178K), positive charge substitutions for H2 ( S186K or S186R, or Q179K or Q179R), and negative charge substitution (Q124E) in L2. Another small cluster was represented by a single member of this small cluster (unique identifier 9049-9759) and additionally contained substitutions for the formation of modified disulfide bonds. The remaining clusters were represented by the unique identifiers 9682-9740, using drivers similar to the other two smaller clusters for H1 and L1, but using different constant region H2 and L2 drivers. H2 utilized L143R or L143K, and L2 utilized V133E or V133D in addition to the Q124E substitution (shared with the other two small clusters).

  For cluster 12, four representative designs (9696-9848, 9986-9978, 9692-9846 and 9587-9735) were each selected to represent four small clusters (see Table 26). All members of this cluster utilized electrostatic substitution to guide preferential pairing of heterodimers. Some members made additional use of solid drivers. The small cluster represented by the unique identifier 9696-9848 utilized both electrostatic and solid drivers. The electrostatic substitution shared in this small cluster is composed of L143E at H1, Q124R and T178R at L1, and similarly located at S186K or S186R, or Q179K or Q179R of H2, and Q124E and T180E of L2. doing. The stereosubstitution shared in this cluster is composed of S188L in H1 and S176L or V133Y or V133W in L2, and in the design utilizing V133Y or V133W in L2, L143A or L124A is also present in H2 and bulky. Has a high mutation. A small cluster represented by the unique identifier 9692-9848 utilizes a similar electrostatic driver compared to a small cluster represented by the unique identifier 9696-9848, and some members have a similar location. The replacement T178E was utilized in L2 instead of T180E. In addition, a subset of this small cluster also utilized a similar stereo driver and used the similarly located substitution T178Y or T178F in L2 instead of S176L. Small clusters represented by the unique identifiers 9986-9978 utilized only one electrostatic driver to guide preferential matching. Similar covalent substitutions were used for H1, L1 and H2, but a different L2 substitution (S131E) was used. The remaining small cluster was represented by the unique identifiers 9587-9735 and utilized electrostatic drivers similar to H1 and L1, except that L1's T178R was not used by all members in this small cluster. Different electrostatic clusters were utilized in H2 (L143R or L143K) and L2 (Q124E and V133E or Q124E and V133D). A small number of members in this small cluster also utilized similar solid drivers composed of S188L at H1 and S176L at L2. Overall, in preferentially paired heterodimers, charge substitution in the Fab region allows the formation of salt bridges. In mismatched heterodimers, formation is rejected by electrostatic repulsion. Furthermore, in designs that also utilize solid drivers, formation is additionally rejected due to solid effects. Cluster 13 is composed of one member 9122-9371 (see Table 27). This design utilized a modified disulfide as a covalent driver for preferential pairing of heterodimers between H1 F122C and L1 Q124C. In addition, since the design lacks natural interchain disulfides, the formation of disulfide bonds was confirmed by non-reducing and reducing SDS-PAGE gels. This design was not tested in the SMCA format, but the modified disulfide was tested in combination with an additional constant region driver in the presence of natural interchain disulfide (Cluster 3, representative designs 9066-9335).

Transfection method A co-expression set comprising two heavy chain and two light chain constructs was transfected into CHO-3E7 cells as follows. CHO-3E7 cells were cultured in FreeStyle ™ F17 medium (Invitrogen catalog number A-1383501) supplemented with 4 mM glutamine and 0.1% Pluronic F-68 (Invitrogen catalog number 24040-032) from 1.7 to The cells were cultured at 37 ° C. at a density of 2 × 10 ⁶ cells / ml. A total volume of 50 ml was transfected with a total of 50 μg of DNA at a DNA: PEI ratio of 1: 2.5 by using PEI-pro (Polyplus catalog number 115-010). Twenty-four hours after adding the DNA-PEI mixture, the cells were transferred to 32 ° C. and incubated for 7 days before harvesting. The culture medium was collected by centrifugation and vacuum filtered using a Sterilip 0.2 μM filter. The filtered culture medium was then purified as follows by use of Protein A MabSelect SuRe resin (GE Healthcare number 17-5438-02). The filtered culture medium was applied to a column (Hyclone DPBS / modified, no calcium, no magnesium, number SH-300028.02) previously equilibrated with PBS. The heterodimeric antibody species was then washed with PBS and eluted with Amicon ultra 15 centrifugal filter Ultracel 10K (Millipore number SCGP00525) with 100 mM citrate pH 3.6. The buffer was then exchanged with PBS and samples were evaluated by calipers prior to deglycosylation and LC-MS.

  To assess the bispecific bias inherent in the wild type bispecific Ab system, where one system of light chains preferentially binds to both Ab heavy chains, H1: H2: L1: A set of L2 DNA ratios was tested in CHO expression. These ratios attempt to compensate for natural differences in the heavy and light chain expression levels of two different antibodies and / or inherent pairing bias. In all bispecific Ab systems, deflection was observed across all ratios tested (Figure 9). In the D3H44 / trastuzumab system, deflection is observed towards trastuzumab, ie, the D3H44 heavy chain preferentially pairs with the trastuzumab light chain (see FIG. 9a). For D3H44 / cetuximab, deflection is observed towards cetuximab, ie, the D3H44 heavy chain preferentially pairs with the cetuximab light chain (see FIG. 9b). In the trastuzumab / cetuximab system, deflection is observed towards trastuzumab, ie, the cetuximab heavy chain preferentially pairs with the trastuzumab light chain (see FIG. 9c).

  In a test in each of the 25 representative designs in each bispecific Ab system, the H1: H2: L1: L2 DNA ratio used yielded the highest amount of bispecific Ab species and at the same time low amounts From the corresponding wild-type bispecific system with half Ab (see Tables 32a, b and c). In the D3H44 / trastuzumab system, the ratios used are 15 (H1), 15 (H2), 53 (L1), 17 (L2), where H1 and L1 refer to D3H44, where H2 and L2 are See trastuzumab. For the D3H44 / cetuximab system, the ratios used are 15 (H1), 15 (H2), 17 (L1), 53 (L2), where H1 and L1 refer to trastuzumab and H2 and L2 are Reference is made to cetuximab. For the D3H44 / cetuximab system, the ratios used are 15 (H1), 15 (H2), 53 (L1), 17 (L2), where H1 and L1 refer to D3H44, where H2 and L2 are Reference is made to cetuximab.

  Further, substitutions present in H1L1 and H2L2 in one orientation were tested in bispecific Ab-based antibody 1 (Ab1) and antibody 2 (Ab2), respectively, and in the other “inverted” orientation, H1L1 and The design tested the D3H44 / cetuximab and trastuzumab / cetuximab bispecific systems in both orientations so that substitutions present in H2L2 were tested in Ab2 and Ab1, respectively (see Tables 28a and b). “_1” attached to the unique identifier indicates that the heavy chain and associated light chain substitution (see Table 13a) gives a stronger LCCA preferential pairing result, but the heavy chain is compared to the light chain of other antibodies. Thus refer to a design placed on an antibody that weakly competes with the associated light chain. “_2” attached to the unique identifier indicates that the heavy chain and associated light chain substitution (see Table 13a) gives a stronger LCCA preferential pairing result, but the heavy chain is compared to the light chain of other antibodies. Refers to the opposite “inverted” orientation placed on the antibody that weakly competes with the associated light chain. For the D3H44 / trastuzumab system, the design was tested only in the “_1” orientation (see Table 28c).

SMCA Results The D3H44 / trastuzumab system was treated with only one enzyme (PNGase F) and completely deglycosylated. In multi-enzyme treatment, the sugar bound to the Fab region was generally cleaved to either core M3F (using 4 enzyme treatments) or G0F (using 3 enzymes). Overall, in most cases, the deglycosylation process provided the ability to identify all possible different species identified by LC-MS. In many cases, each species was represented by a single LC-MS peak. Exceptions include side peaks (possibly heterogeneity due to cleavage of the adduct or leader peptide) that may correspond to the desired bispecific species, but due to subpeak ambiguity Thus, these sub-peaks were not considered for their contribution to bispecific species. In addition, some designs in the D3H44 / cetuximab (3519_1, 3522_1) and trastuzumab / cetuximab (9748-9338_1) systems require multiple peaks to account for the species due to the variability of the combined high mannose. did. All of these designs introduced glycosylation sites in the cetuximab light chain. In some cases, due to the low mass separation between species (ie, less than 50 Da difference), it is not possible to distinguish some minor species (constituting less than 5% of all species) It was. Furthermore, the desired bispecific species H1-L1_H2-L2 is generally not experimentally distinguishable from mispaired H1-L2_H2-L1 based on LC / MS. Thus, when bispecific content is reported in the table, it cannot be completely ruled out that it does not contain this type of mismatched species. However, the very low content observed in species such as H1-L2_H1-L2 and H2-L1_H2-L1, and H1-L2 and H2-L1 half antibodies is very high, if any, of bispecific species. It shows that there is less pollution.

LC-MS data is presented in Tables 29a, 29b and 29c. For comparison, wild-type data is also shown in Tables 33a, 33b, and 33c, and “NA” is shown in the “SMCA unique identifier” column and the “cluster” column. All three bispecific wild type systems showed a biased bias so that one light chain preferentially binds both heavy chains (see Table 33 and FIG. 9). Furthermore, at least in the trastuzumab / cetuximab system, tag placement also appears to have a significant effect on H1L1 and H2L2 pairings. Therefore, to evaluate the effect of the design on the transferability and rate of the desired bispecific species relative to the wild type, at the same H1: H2: L1: L2 DNA ratio with the corresponding wild type bispecific construct. Comparison of "H1L1 pairing ratio (%) relative to wild type (for all H1 species)", "H2L2 pairing ratio (%) compared to wild type (for all H2 species) ”And“ Change in H1: H2: L1: L2 ratio (%) relative to wild type ”(considering only full-size antibody species) (see Table 29). In either assessment of H1L1 pairing percentage (%) (for all H1 species) or H2L2 pairing percentage (%) (all for H2 species), all species are assessed for pairing in the Fab region. If the corresponding wild-type bispecific species antibody was not evaluated by SMCA, the comparison was done in a similar wild-type construct. The estimate is indicated by “ ^*** ” immediately adjacent to the reported value. Similar wild type constructs selected for comparison were selected as follows. To assess transferability, each wild-type construct was given the highest “% H1L1 pairing percentage (%) and H2L2 pairing percentage (%) for all species” experiments (different ratios). Performed). To assess the effect of the design on the percentage of desired bispecific species relative to wild type, each wild type construct was expressed as the highest H1: H2: L1: L2 ratio (% full size antibody species only). SMCA experiment (performed at different ratios). In both cases, among all wild type constructs in the bispecific species, the median value was selected as the comparative wild type value.

  In each design, the transferability is determined by the ratio of H1L1 / all H1 species and / or H2L2 / all H2 species in the overall H: L pairing across all species with respect to WT ( %) Was assessed by noting the increase in In addition, the effect on the rate of the desired bispecific species is also evaluated, since half antibodies can be removed / minimized by preparative SEC, if present, or by further H1: H2: L1: L2 DNA titration. The comparison of only full size antibody species is emphasized. Tables 30a, b and c show that preparative SEC can be effective in removing / minimizing semi-Ab species. Tables 32a, b and c show that the rate of half-Ab species can also be manipulated during transfection using various DNA titration ratios.

  In the D3H44 / cetuximab system (Table 29a), all but one design (9327-6054_2) was migrated as assessed by H1L1 pairing across all species for wild type. Most designs (except 9327-6054_2 and 9134-9521_2) also showed an increase in the desired bispecific antibody rate when considering only the complete Ab species. In addition, except for one design that did not migrate (9327-6054_2), the design reduced the main mismatched antibody species (H1H2L2L2) observed in the wild type. In addition, with the exception of 9327-6054_2 and the corresponding 9327-6054_1 in the other orientation, the design is transitioned in both orientations, and most designs have similar effective H: L matching in both orientations. Indicated.

  In the D3H44 / trastuzumab system (Table 29b), all designs were migrated as assessed by H1L1 pairing across all species for wild type. In addition, all designs showed an increase in the desired bispecific antibody rate (when considering only full Ab species). Furthermore, most designs significantly reduced the main mismatched antibody species (H1H2L2L2) observed in the wild type. However, no data was reported for 9611-9077_1 due to lack of expression (Table 28c).

  In the trastuzumab / cetuximab system (Table 29c) at least 35 out of 49 designs evaluated H2L2 pairing across all H2 species and showed migratory potential ("change in% H2L2 pairing relative to wild type" (Positive values in the column “for all H2 species”). Designs that may not have been migrated include 9279-9518_2, 3522_2, 9815-9825_2, 9327-6054_2, 9118-6098_2, 9748-9338_2, 9692-9846_2, 9587-9735_2, 9814-9828_2, 3519_2, 9986-9978_2, 9168-9342_2 and 9066-9335_1 are included (negative values in column of “change in% H2L2 vs. wild type (%) for all H2 species”), but the design of the other orientation can be migrated Sex was not inhibited (note that 9279-9518_1 was not tested because it lacked the sample). All designs that showed migratory potential showed a decrease in the rate of the main mismatched antibody species (H1H2L1L1) observed in wild type experiments. In addition, of the migrated designs, only two designs (9134-9521_1 and 9279-9518_2) achieved the desired bispecific Ab rate when considering only the complete antibody species compared to the wild type. Showed a decrease.

  In general, most designs that increased H: L pairing of weak competing antibodies resulted from an increased rate of the desired bispecific species (considering only full size antibodies). With respect to orientation, most designs showed “_1” orientation, where “_2” showed a similar or better transition potential compared to H: L pairing compared to transition (exception is trastuzumab / cetuximab It was mainly observed in the system). In addition, Table 35a covers designs that migrated in both orientations in all three bispecific systems tested (D3H44 / cetuximab, D3H44 / trastuzumab and trastuzumab / cetuximab). Table 35b shows the design transferred in one orientation in all three bispecific systems tested (D3H44 / cetuximab, D3H44 / trastuzumab and trastuzumab / cetuximab) and the other orientation in the only bispecific system The design that was migrated in is covered. In addition, in a particular orientation, the same mutation is present in the heavy chain of the three bispecific systems and the weakly competing cognate light chain, with light chain utilization exceeding at least 10%.

  With regard to cluster transferability and performance, in the D3H44 / trastuzumab bispecific system, all members in all clusters exhibit transferability (see FIG. 11a) and the desired bispecific antibody. The rate of (considering only full size antibodies) was increased (see FIG. 11b). In the D3H44 / cetuximab bispecific system, all clusters showed migratory potential and only one member in cluster 3 showed a decrease in H1L1 pairing across all H1 species compared to wild type ( See FIG. 11c). In addition, all clusters contained members that showed an increase in the desired bispecific antibody rate relative to wild type (considering only full-length antibodies), while the three clusters (clusters 1, 3 and 4) were Also included were members who showed a decrease in the desired bispecific antibody ratio (considering full-length antibody only) relative to wild type (see Table 11d). For the trastuzumab / cetuximab bispecific system, all clusters contain variants that exhibit the desired migratory potential, but only a few clusters (1, 5, 7, 8, 10, 11) have corresponding members All include variants that showed migratory potential (see FIG. 11e). In addition, all clusters contain members that show an increase in the desired bispecific antibody rate (considering only full size antibodies) with respect to the wild type (see FIG. 11f). In clusters where all members showed migratory potential, all members in clusters 5, 7, 8, 10 and 11 increased to the desired bispecific antibody rate (considering only full size antibodies) with respect to wild type Also shown.

  Overall, when all three bispecific systems are considered together, all members in clusters 1, 5, 7, 8, 10 and 11 show migratory potential (see FIG. 11g) Clusters 5, 7, 8, 10 and 11 contained members where all members showed an increase in the desired bispecific antibody rate (considering only full size antibodies) relative to the wild type (see FIG. 11h). To do).

  Overall, most designs that increased H: L pairing of weak competing antibodies resulted from an increased rate of the desired bispecific species (considering only full size antibodies). With respect to orientation, most designs showed “_1” orientation, where “_2” showed a similar or better transition potential compared to H: L pairing compared to transition (exception is trastuzumab / cetuximab It was mainly observed in the system).

Example 10 Preparative Size Exclusion Chromatography (SEC) of SMCA Bispecific Heterodimeric Antibody and Parent Mab Selected in Biophysical Characterization
A subset of SMCA samples was selected for additional biophysical characterization. Most of these SMCA samples typically show high pairings (H1L1 + H2L2 / more than about 80% in all species tandem) and low amounts of half-antibody species (about all half-antibody species Less than 30%). Preparative SEC was performed as follows. Heterodimeric antibody samples were separated by use of a Superdex 200 10/300 GL (GE Healthcare) column attached to a Pharmacia (GE Healthcare) AKTA Purifier system. A heterodimeric antibody sample (0.3-0.5 ml) in PBS (Hyclone DPBS / modified, no calcium, no magnesium, catalog number SH-300028.02) was placed in a 0.5 ml loop filled with PBS. Loaded manually. The sample was then automatically injected into the column and degraded at 0.5 ml / min with a 1 CV elution volume. Protein elution was monitored at OD ₂₈₀ and collected in 0.5 ml fractions. In each SMCA sample, these fractions containing the main peak were pooled and further biophysically characterized.

Example 11 Evaluation of Preferential Pairing of Bispecific Heterodimers in Antibody Format After Preparative Size Exclusion Chromatography After preparative SEC, selected samples were described in Example 9. Preferential pairing of bispecific heterodimeric antibodies was analyzed by using LC-MS method. All these samples increase the rate of the desired bispecific antibody species and decrease the rate of half-antibody species (Tables 29 and 30).

Example 12: Thermal stability of SMCA bispecific heterodimeric antibodies After preparative SEC, the thermal stability of selected SMCA bispecific heterodimeric antibodies was determined to determine the parental D3H44 and trastuzumab monoclonals. The antibody was compared to the cetuximab 1 arm antibody. In general, a one-arm antibody is composed of one full-length heavy chain, one truncated heavy chain that lacks the Fab region (and incorporates a C233S substitution), and one light chain, as described in Example 9. Refers to a construct with heavy chain heterodimerization achieved as described.

Measurement of Thermal Stability The thermal stability of selected bispecific heterodimeric antibodies and wild type controls was measured using differential scanning calorimetry (DSC) as follows. After preparative SEC treatment, 400 μL samples were mainly used for DSC analysis by VP-Capillary DSC (GE-Healthcare) at a concentration of 0.2 mg / ml or 0.4 mg / mL in PBS. At the beginning of each DSC run, five buffer blank injections were performed to stabilize the baseline and buffer injections were made prior to each sample injection for reference. Each sample was scanned at a rate of 60 ° C./hour at 20-100 ° C., low feedback, 8 seconds filter, 5 minutes preTstat and 70 psi nitrogen pressure. The obtained thermogram was used as a reference and analyzed using Origin 7 software.

  The results are shown in Tables 31a, b and c. The Fab Tm values reported in the table for the wild type were obtained from D3H44 (79 ° C.) and trastuzumab (81 ° C.) homodimeric antibodies, and from cetuximab (72 ° C.) 1-arm antibodies. For the WT D3H44 / cetuximab and trastuzumab / cetuximab heterodimeric antibodies, only two peaks corresponding to Fab Tm are observed. A separate peak is not observed in CH2 (due to overlap with cetuximab Fab) or in CH3 (due to overlap in Tm values of D3H44 and trastuzumab Fab). For the WT D3H44 / trastuzumab heterodimeric antibody, the Tm values of the two Fabs of D3H44 and trastuzumab are similar, so the peak at 81 ° C. may correspond to both Fabs, while approximately 72 ° C. The peak at may correspond to CH2.

  In Tables 31a, b and c, only the Tm values for the peaks corresponding to both Fabs were reported unless otherwise indicated. Note also that in some heterodimeric samples, the protein concentration was low (less than 0.4 mg / mL), resulting in increased noise at baseline. As a result, in the D3H44 / trastuzumab system, some samples produced low peak intensity DSC curves that were difficult to distinguish CH2 peaks and could destabilize the Fab. In this case, Tm values of 70-72 ° C. were also reported (Table 31a). Overall, most heterodimeric antibodies show thermal stability (below 3 ° C.) similar to the corresponding wild-type molecule. Furthermore, most heterodimers do not show additional peaks suggesting significant instability of the CH2 or CH3 peaks. One exception includes the modified heterodimeric antibody 9611-9077_2 of the trastuzumab / cetuximab heterosystem, which shows an additional peak at 60 ° C. that can be attributed to CH2 destabilization.

Example 13: Measurement of antigen affinity of a bispecific heterodimeric antibody The ability of a bispecific antibody to bind to the relevant antigen determines whether amino acid substitutions have any effect on antigen binding. Evaluated for. Antigen binding affinity was determined by SPR as follows.

SPR biosensor assay EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, NHS: N-hydroxysuccinimide, SPR: surface plasmon resonance, EDTA: ethylenediaminetetraacetic acid, TF: tissue factor, EGFR ECD : Epidermal growth factor receptor extracellular domain, Her2 ECD: Human epidermal growth factor receptor 2 extracellular domain.

  SPR supply. Series S Sensor Chip CM5, Biacore amine coupling kit (NHS, EDC and 1M ethanolamine), and 10 mM sodium acetate buffer were purchased from GE Healthcare Life Science (Mississauga, ON). Recombinant Her2 extracellular domain (ECD) protein was purchased from eBioscience (San Diego, Calif.). PBS running buffer with 1% Tween 20 (PBST) is available from Taknova Inc. Goat polyclonal human Fc antibody purchased from (Hollister, Calif.) Is available from Jackson Immuno Research Laboratories Inc. (West Grove, PA). EDTA was purchased from Bioshop (Burlington, ON).

  All surface plasmon resonance assays were performed using a Biacore T200 Surface Plasson Resonance instrument (GE Healthcare Life Science, at a temperature of 25 ° C. with PBST running buffer (0.5 M EDTA stock solution added to a final concentration of 3.4 mM). (Mississauga, ON)). The anti-human Fc capture surface was generated using the Series S Sensor Chip CM5 using default parameters under Immobilization Wizard with Biacore T200 control software set to target 2000 resonance units (RU). Selection of antibody variants that bind to the Her2 ECD, TF or EGFR ECD antigen target was performed in two steps. Indirect capture of antibody variants to the anti-human Fc antibody flow cell surface followed by injection of 5 concentrates of purified antigen for kinetic analysis using a single cycle kinetic methodology. Variants or controls were injected at 1 μg / mL into individual flow cells for 60 seconds at a flow rate of 10 μL / min for capture. In general, this results in capture of approximately 50-100 RU on the anti-human Fc surface. The first flow cell was left empty and used as a blank control. Immediately following this capture step, five concentrations of antigen (5 nM, 2.5 nM, 1.25 nM, 0.63 nM and 0.31 nM for TF or EGFR ECD antigen, or 40 nm, 20 nm, 10 nm, 5 nm for Her2 ECD antigen And 2.5 nm) were injected sequentially over all four flow cells at 100 μL / min for 180 seconds with a dissociation phase of 300 seconds for EGFR ECD, 1800 seconds for Her2 ECD and 3600 seconds for TF. The capture antibody surface was regenerated for 120 seconds at 30 μL / min with 10 mM glycine, pH 1.5, and prepared for the next injection cycle. At least two mock buffer injections were performed for each analyte injection used as a reference. The resulting single cycle kinetic sensorgram was analyzed by use of Biacore T200 BiaEvaluation software and fitted to a 1: 1 binding model.

  The antigen affinity of the heterodimeric antibody was assessed with reference to the corresponding wild type controls: D3H44, trastuzumab OAA and cetuximab OAA Mabs. Although antigen affinity was also obtained from wild-type bispecific antibodies, WT bispecific SPR capture can be heterogeneous (eg, with capture of mismatched heterodimers) Prevents determination of KD (see Tables 31a and c). For heterodimeric antibodies with antigen binding measured in the D3H44 / cetuximab system, antigen affinity was similar to the corresponding WT control (see Table 31b). For most heterodimeric antibodies with antigen binding measured in both the D3H44 / trastuzumab and trastuzumab / cetuximab systems, antigen affinity was similar to the corresponding WT control (see Tables 31a and c) about). Exceptions include 11 modified antibodies that did not show Her2 binding. In both the D3H44 / trastuzumab and trastuzumab / cetuximab systems, Her2 binding was not observed in the six modified heterodimeric antibodies 9049-9759_1 and 9682-9740_1 and 3522_1. In addition, in the trastuzumab / cetuximab system, five further modified antibodies 9696-9848_1, 9561-9095_2, 9611-9077_2, 9286-9402_2 and 9060-9756_2 also lacked binding to Her2. Ten of these eleven modified antibodies shared constant region mutations in the heavy chain (L143E_K145T) and light chain (Q124R_T178R). The other engineered antibody 9286-9402_2 shared the same constant region mutation in the heavy chain (L143E_K145T) and a mutation similar to the light chain (Q124K and S176R).

Example 14: Ultra High Performance Liquid Chromatography Size Exclusion Chromatography (UPLC-SEC) Profile of Modified Heterodimeric Antibodies and Wild Type Heterodimers and Homodimer Antibodies Modified Heterodimeric Antibodies and Control Wild After preparative SEC of type bispecific and homodimeric antibodies, a Waters BEH200 SEC column (2.5 mL, 4.6 × 150 mm) set at 30 ° C. and attached to a Waters Accuracy UPLC system with a PDA detector. , Stainless steel, 1.7 μm particles) was used to perform UPLC-SEC. The run time consisted of 7 minutes and the total volume per injection was 2.8 mL with running buffer PBS and 0.02% polysorbate 20 or 20 mM NaPO4, 50 mM KCl, 0.02% polysorbate 20 10% acetonitrile, pH 7, was used at 0.4 ml / min. Elution was monitored by UV absorption ranging from 210 to 400 nm and the chromatogram was extracted at 280 nm. Peak integration was performed using Empower3 software.

  FIG. 10 shows UPLC-SEC profiles of a representative modified heterodimeric antibody and a representative WT heterodimer. In most cases, the modified heterodimeric antibody exhibits a UPLC-SEC profile similar to the corresponding WT heterodimeric antibody, with the average percentage of monomer in each of D3H44 / trastuzumab, D3H44 / cetuximab and trastuzumab / cetuximab Were 99.18%, 98.70% and 98.77% (see Tables 31a, 31b and 31c).

  Although the present invention has been shown and described with reference to preferred and various alternative embodiments, various changes in form and detail can be made without departing from the spirit and scope of the invention, It will be understood by those skilled in the art.

  All references, issued patents, patent publications and sequence accession numbers disclosed herein are hereby incorporated by reference in their entirety for all purposes.

Table Keys Table 1: Main criteria for Fab model Table 2: Hot spot amino acid positions at the heavy and light chain interface of D3H44 (a typical Fab containing kappa light chain) Table 3A. Kabat numbering table for heavy chain amino acid sequences of D3H44, trastuzumab and cetuximab 3B. Kabat numbering of light chain amino acid sequences of D3H44, trastuzumab and cetuximab Table 3C: Amino acid and DNA sequence of D3H44, trastuzumab and cetuximab 4. LCCA design table with modifications that preferentially pair H1 with L1 in one immunoglobulin heavy chain and / or two immunoglobulin light chains. Design library Table 6. Core design table 7. Example of combination design Table 8. Example of modification / optimization design Table 9. Example of combination design including optimization design Table 10. Examples of combinatorial designs including independent designs Table 11: H1: L1: L2 DNA ratio used for light chain competition assay and validation Table 12. Evaluation of LCCA performance, stability and antigen binding of LCCA designs arranged to reduce DSF values of H1L1 Fab heterodimers 13a. Exactly paired Fab heterodimer: LCCA performance table of design meeting LCCA average performance criteria with mispaired Fab heterodimer being 86:14 Table 13b. Exactly paired Fab heterodimers: Design stability and antibody binding assessments meeting LCCA average performance criteria of 86:14 mispaired Fab heterodimers 14a. Exactly paired Fab heterodimer: LCCA performance table of the design for mispaired Fab heterodimers that function below the LCCA average performance criteria of 86:14 Table 14b. Accurately paired Fab heterodimer: Evaluation of stability and antibody binding of a design that works below the LCCA average performance criteria with mispaired Fab heterodimer being 86:14. Cluster 1 design table including representative designs 16. Cluster 2 design table including representative designs 17. Cluster 3 design table including representative designs 18. 18. Cluster 4 design table including representative designs 21. Design table for cluster 5 including representative designs 20. Cluster 6 design table including representative designs Cluster 7 design table including representative designs 22. Cluster 8 design table including representative designs 23. Cluster 9 design table including representative designs 24. Cluster 10 design table including representative designs 25. Cluster 11 design table including representative designs 26. Cluster 12 design table 27 including representative designs 27. Cluster 13 design table 28a. SMCA specific identifier of trastuzumab / cetuximab bispecific system Table 28b. SMCA unique identifier of D3H44 / cetuximab bispecific system Table 28c. SMCA-specific identifier of the D3H44 / trastuzumab bispecific system Table 29a. LC-MS pairing data for heterodimeric antibodies derived from the D3H44 (H1L1) / cetuximab (H2L2) bispecific system and yield after pA (mg / L)
Table 29b. LC-MS pairing data and yield (mg / L) of heterodimeric antibody derived from D3H44 (H1L1) / trastuzumab (H2L2) bispecific system and pA
Table 29c. LC-MS pairing data and yield after pA (mg / L) of heterodimeric antibodies derived from the trastuzumab (H1L1) / cetuximab (H2L2) bispecific system
Table 30a. LC-MS pairing table of heterodimeric antibodies derived from D3H44 (H1L1) / cetuximab (H2L2) bispecific system after preparative SEC Table 30b. LC-MS pairing table of heterodimeric antibodies derived from D3H44 (H1L1) / trastuzumab (H2L2) bispecific system after preparative SEC 30c. LC-MS Pairing Table of Heterodimeric Antibodies from Trastuzumab (H1L1) / Cetuximab (H2L2) Bispecific System After Preparative SEC 31a. Biophysical characterization of selected designs derived from the D3H44 / trastuzumab system (antigen binding, thermal stability, UPLC-SEC)
Table 31b. Biophysical characterization of selected designs derived from the D3H44 / cetuximab system (antigen binding, thermal stability, UPLC-SEC)
Table 31c. Biophysical characterization of selected designs derived from the trastuzumab / cetuximab system (antigen binding, thermal stability, UPLC-SEC)
Table 32a. Effect of DNA titration ratio of wild type D3H44 / trastuzumab system on the percentage of antibody species as assessed by LC-MS. H1 and L1 refer to the heavy and light chains of D3H44, respectively. H2 and L2 refer to the heavy and light chains of trastuzumab, respectively.
Table 32b. Effect of DNA titration ratio of wild type D3H44 / cetuximab system on the percentage of antibody species as assessed by LC-MS. H1 and L1 refer to the heavy and light chains of D3H44, respectively. H2 and L2 refer to the heavy and light chains of cetuximab, respectively.
Table 32c. Effect of DNA titration ratio of wild-type trastuzumab / cetuximab system on the percentage of antibody species as assessed by LC-MS. H1 and L1 refer to the heavy and light chains of trastuzumab, respectively. H2 and L2 refer to the heavy and light chains of cetuximab, respectively.
Table 33a. LC-MS pairing of wild-type antibody constructs derived from the D3H44 (H1L1) / cetuximab (H2L2) bispecific system Table 33b. LC-MS pairing table of wild-type antibody constructs derived from the D3H44 (H1L1) / trastuzumab (H2L2) bispecific system Table 33c. LC-MS pairing table of wild-type antibody constructs derived from the trastuzumab (H1L1) / cetuximab (H2L2) bispecific system Stabilization of mutations in Fab heterodimers Table 35a. Design table 35b showing mobility in both orientations in all three bispecific systems (D3H44 / cetuximab, D3H44 / trastuzumab and trastuzumab / cetuximab). The mobility in all three bispecific systems (D3H44 / cetuximab, D3H44 / trastuzumab and trastuzumab / cetuximab) is shown in one orientation and migrates to the other orientation only in one bispecific system and simultaneously light Designs that meet at least 10% of the chain utilization criteria

(Table 1) Main criteria for the Fab model

*Ｋａｂａｔ番号付け TABLE 2 Hot spot amino acid positions at the heavy and light chain interface of D3H44 (a typical Fab containing a kappa light chain)

* Kabat numbering

Table 3A: Kabat numbering of heavy chain amino acid sequences of D3H44, trastuzumab and cetuximab

Table 3B: Kabat numbering of light chain amino acid sequences of D3H44, trastuzumab and cetuximab

Table 3C: Amino acid and DNA sequences of D3H44, trastuzumab and cetuximab

*Ｋａｂａｔ番号付け；ＷＴは、アミノ酸突然変異を有さない野性型免疫グロブリン鎖を指す。
**Ｈ１、Ｌ１及びＬ２突然変異それぞれの特有のセット（ＬＣＣＡフォーマット）が、セット番号または「特有の識別子」として指定された。 Table 4 LCCA design with modifications that preferentially pair H1 with L1 in one immunoglobulin heavy chain and / or two immunoglobulin light chains

* Kabat numbering; WT refers to wild-type immunoglobulin chain without amino acid mutations.
** A unique set (LCCA format) of each of the H1, L1 and L2 mutations was designated as a set number or “unique identifier”.

*Ｋａｂａｔ番号付け；ＷＴは、アミノ酸突然変異を有さない野性型免疫グロブリン鎖を指す。
**「特有の識別子」は、２つの構成要素ＬＣＣＡの特有の識別子から構成される、またはＳＭＣＡフォーマットのみにより試験された設計の単一識別子から構成される。 (Table 5) Design library

* Kabat numbering; WT refers to wild-type immunoglobulin chain without amino acid mutations.
** "Unique identifier" consists of two component LCCA unique identifiers or a single identifier of a design that has been tested by SMCA format only.

*Ｋａｂａｔ番号付け；ＷＴは、アミノ酸突然変異を有さない野性型免疫グロブリン鎖を指す。
**「特有の識別子のセット」は、２つの構成要素ＬＣＣＡの特有の識別子から構成される。 (Table 6) Core design

* Kabat numbering; WT refers to wild-type immunoglobulin chain without amino acid mutations.
** "Set of unique identifiers" consists of the unique identifiers of two components LCCA.

*Ｋａｂａｔ番号付け (Table 7) Example of combination design

* Kabat numbering

*Ｋａｂａｔ番号付け (Table 8) Example of modification / optimization design

* Kabat numbering

(Table 9) Example of combination design including optimization design

*Ｋａｂａｔ番号付け；ＷＴは、アミノ酸突然変異を有さない野性型免疫グロブリン鎖を指す。 (Table 10) Example of combination design including independent design

* Kabat numbering; WT refers to wild-type immunoglobulin chain without amino acid mutations.

^追加のＤＮＡ：ＡＫＴｄｄ ｐＴＴ２２は、構成的に活性なタンパク質キナーゼＢ突然変異体（有意な陽性ＡＫＴ突然変異体）を含有するベクターを指す。ｓｓＤＮＡは、サケ精子ＤＮＡを指す。 TABLE 11 H1: L1: L2 DNA ratio used for light chain competition assay and validation

^ Additional DNA: AKTdd pTT22 refers to a vector containing a constitutively active protein kinase B mutant (significant positive AKT mutant). ssDNA refers to salmon sperm DNA.

*結合Ｌ鎖タグ（ＨＡまたはＦＬＡＧ）の存在／不在のみが異なる他のＦａｂヘテロ二量体から導かれた推定値を示す。
**３３３（Ｈ１）、２５０（Ｌ１）、７４９（Ｌ２）のＬＣＣＡ実験から導かれた値。
***３３３（Ｈ１）、１００（Ｌ１）、８９９（Ｌ２）のＬＣＣＡ実験から導かれた値。
ＮＤは、利用可能なデータがないことを示す。 Table 12: Evaluation of LCCA performance, stability and antigen binding of LCCA designs installed to reduce DSF values of H1L1 Fab heterodimers

* Shows estimates derived from other Fab heterodimers that differ only in the presence / absence of bound light chain tag (HA or FLAG).
** Values derived from LCCA experiments at 333 (H1), 250 (L1), 749 (L2).
*** Values derived from LCCA experiments at 333 (H1), 100 (L1), and 899 (L2).
ND indicates that no data is available.

*値は、Ｌ１：Ｌ２のＤＮＡ比１：３で実施したＬＣＣＡ実験から得て、Ｌ１：Ｌ２のＤＮＡ比を１：１に正規化した。
**値は、Ｌ１：Ｌ２のＤＮＡ比１：９で実施したＬＣＣＡ実験から得て、Ｌ１：Ｌ２のＤＮＡ比を１：１に正規化した。
***「特有の識別子」は、（Ｈ１Ｌ１Ｌ２のセット番号−Ｈ２Ｌ２Ｌ１のセット番号）または（Ｈ２Ｌ２Ｌ１のセット番号−Ｈ１Ｌ１Ｌ２のセット番号）の配向のいずれかの２つの構成要素ＬＣＣＡの特有の識別子からなる。 Table 13a: Correctly paired Fab heterodimer: LCCA performance of a design that meets the LCCA average performance criteria of 86:14 mispaired Fab heterodimer

* Values were obtained from LCCA experiments performed at a L1: L2 DNA ratio of 1: 3 and the L1: L2 DNA ratio was normalized to 1: 1.
** Values were obtained from LCCA experiments performed with a L1: L2 DNA ratio of 1: 9 and the L1: L2 DNA ratio was normalized to 1: 1.
*** "Unique identifier" consists of the unique identifier of the two components LCCA of either (H1L1L2 set number-H2L2L1 set number) or (H2L2L1 set number-H1L1L2 set number) orientation .

*結合Ｌ鎖タグ（ＨＡまたはＦＬＡＧ）の存在／不在のみが異なる他のＦａｂヘテロ二量体から導かれた推定値を示す。
**「特有の識別子」は、（Ｈ１Ｌ１Ｌ２のセット番号−Ｈ２Ｌ２Ｌ１のセット番号）または（Ｈ２Ｌ２Ｌ１のセット番号−Ｈ１Ｌ１Ｌ２のセット番号）の配向のいずれかの２つの構成要素ＬＣＣＡの特有の識別子からなる。 Table 13b: Accurately matched Fab heterodimer: Evaluation of design stability and antibody binding to meet LCCA average performance criteria with mispaired Fab heterodimer of 86:14

* Shows estimates derived from other Fab heterodimers that differ only in the presence / absence of bound light chain tag (HA or FLAG).
** "Unique identifier" consists of the unique identifier of the two component LCCA, either (H1L1L2 set number-H2L2L1 set number) or (H2L2L1 set number-H1L1L2 set number) orientation.

*値は、Ｌ１：Ｌ２のＤＮＡ比１：３で実施したＬＣＣＡ実験から得て、Ｌ１：Ｌ２のＤＮＡ比を１：１に正規化した。
**値は、Ｌ１：Ｌ２のＤＮＡ比１：９で実施したＬＣＣＡ実験から得て、Ｌ１：Ｌ２のＤＮＡ比を１：１に正規化した。
***「特有の識別子」は、（Ｈ１Ｌ１Ｌ２のセット番号−Ｈ２Ｌ２Ｌ１のセット番号）または（Ｈ２Ｌ２Ｌ１のセット番号−Ｈ１Ｌ１Ｌ２のセット番号）の配向のいずれかの２つの構成要素ＬＣＣＡの特有の識別子からなる。 Table 14a: Accurately paired Fab heterodimer: LCCA performance of a design that works below the LCCA average performance criteria with a mispaired Fab heterodimer of 86:14

* Values were obtained from LCCA experiments performed at a L1: L2 DNA ratio of 1: 3 and the L1: L2 DNA ratio was normalized to 1: 1.
** Values were obtained from LCCA experiments performed with a L1: L2 DNA ratio of 1: 9 and the L1: L2 DNA ratio was normalized to 1: 1.
*** "Unique identifier" consists of the unique identifier of the two components LCCA of either (H1L1L2 set number-H2L2L1 set number) or (H2L2L1 set number-H1L1L2 set number) orientation .

*結合Ｌ鎖タグ（ＨＡまたはＦＬＡＧ）の存在／不在のみが異なる他のＦａｂヘテロ二量体から導かれた推定値を示す。
**「特有の識別子」は、（Ｈ１Ｌ１Ｌ２のセット番号−Ｈ２Ｌ２Ｌ１のセット番号）または（Ｈ２Ｌ２Ｌ１のセット番号−Ｈ１Ｌ１Ｌ２のセット番号）の配向のいずれかの２つの構成要素ＬＣＣＡの特有の識別子からなる。 Table 14b: Correctly paired Fab heterodimer: Evaluation of stability and antibody binding of a design that functions below the LCCA average performance criteria with a mispaired Fab heterodimer of 86:14

* Shows estimates derived from other Fab heterodimers that differ only in the presence / absence of bound light chain tag (HA or FLAG).
** "Unique identifier" consists of the unique identifier of the two component LCCA, either (H1L1L2 set number-H2L2L1 set number) or (H2L2L1 set number-H1L1L2 set number) orientation.

*Ｋａｂａｔ番号付け
**代表的な設計 (Table 15) Cluster 1 design including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計 (Table 16) Cluster 2 design including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計 (Table 17) Cluster 3 design including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計 (Table 18) Cluster 4 design including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計 Table 19: Design of cluster 5 including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計
***代表的な設計は、Ｌ２がＱ３８Ｅ置換を欠いていることを除いて、９７４５−９０７５に類似している。 Table 20: Cluster 6 design including representative designs

* Kabat numbering
** Representative design
*** A typical design is similar to 9745-9075 except that L2 lacks the Q38E substitution.

*Ｋａｂａｔ番号付け
**代表的な設計 (Table 21) Cluster 7 design including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計 Table 22: Cluster 8 design including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計
***代表的な設計は、Ｌ２がＱ３８Ｅ置換を欠いていることを除いて、９７５１−９０６５に類似している。 (Table 23) Cluster 9 design including representative designs

* Kabat numbering
** Representative design
*** A typical design is similar to 9751-9065 except that L2 lacks the Q38E substitution.

*Ｋａｂａｔ番号付け
**代表的な設計 Table 24: Cluster 10 design including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計 Table 25: Design of cluster 11 including representative designs

* Kabat numbering
** Representative design

*Ｋａｂａｔ番号付け
**代表的な設計 Table 26: Design of cluster 12 including representative designs

* Kabat numbering
** Representative design

(Table 27) Cluster 13 design including representative designs

*Ｋａｂａｔ番号付け。ＷＴ残基は、Ｄ３Ｈ４４系を指すことに留意すること。 Table 28a: SMCA unique identifier of trastuzumab / cetuximab bispecific system

* Kabat numbering. Note that the WT residue refers to the D3H44 system.

*Ｋａｂａｔ番号付け。ＷＴ残基は、Ｄ３Ｈ４４系を指すことに留意すること。 Table 28b: SMCA unique identifier of D3H44 / cetuximab bispecific system

* Kabat numbering. Note that the WT residue refers to the D3H44 system.

*Ｋａｂａｔ番号付け。ＷＴ残基は、Ｄ３Ｈ４４系を指すことに留意すること。 Table 28c: SMCA unique identifier of D3H44 / trastuzumab bispecific system

* Kabat numbering. Note that the WT residue refers to the D3H44 system.

*完全なＡｂ種のみを考慮した割合（％）
**全ての種を考慮した割合（％）
***野生型に対するＨ１：Ｈ２：Ｌ１：Ｌ２の割合（％）の推定変化 Table 29a: LC-MS pairing data and yield (mg / L) of heterodimeric antibodies derived from D3H44 (H1L1) / cetuximab (H2L2) bispecific system and pA

* Percentage considering only complete Ab species (%)
** Percentage considering all species (%)
*** Estimated change in the ratio (%) of H1: H2: L1: L2 to wild type

*完全なＡｂ種のみを考慮した割合（％）
**全ての種を考慮した割合（％）
***野生型に対するＨ１：Ｈ２：Ｌ１：Ｌ２の割合（％）の推定変化 Table 29b: LC-MS pairing data and yield after pA (mg / L) of heterodimeric antibodies derived from the D3H44 (H1L1) / trastuzumab (H2L2) bispecific system

* Percentage considering only complete Ab species (%)
** Percentage considering all species (%)
*** Estimated change in the ratio (%) of H1: H2: L1: L2 to wild type

*完全なＡｂ種のみを考慮した割合（％）
**全ての種を考慮した割合（％）
***野生型に対する推定変化 Table 29c: LC-MS pairing data and yield (mg / L) of heterodimeric antibodies derived from the trastuzumab (H1L1) / cetuximab (H2L2) bispecific system

* Percentage considering only complete Ab species (%)
** Percentage considering all species (%)
*** Estimated change relative to wild type

*完全なＡｂ種のみを考慮した（％）
**全ての種を考慮した（％）
***野生型に対するＨ１：Ｈ２：Ｌ１：Ｌ２の割合（％）の推定変化 Table 30a: LC-MS pairing of heterodimeric antibodies derived from a D3H44 (H1L1) / cetuximab (H2L2) bispecific system after preparative SEC

* Considering only complete Ab species (%)
** Considering all species (%)
*** Estimated change in the ratio (%) of H1: H2: L1: L2 to wild type

*完全なＡｂ種のみを考慮した（％）
**全ての種を考慮した（％）
***野生型に対するＨ１：Ｈ２：Ｌ１：Ｌ２の割合（％）の推定変化 Table 30b: LC-MS pairing of heterodimeric antibodies derived from D3H44 (H1L1) / trastuzumab (H2L2) bispecific system after preparative SEC

* Considering only complete Ab species (%)
** Considering all species (%)
*** Estimated change in the ratio (%) of H1: H2: L1: L2 to wild type

*完全なＡｂ種のみを考慮した（％）
**全ての種を考慮した（％）
***野生型に対する推定変化 Table 30c: LC-MS pairing of heterodimeric antibodies derived from trastuzumab (H1L1) / cetuximab (H2L2) bispecific system after preparative SEC

* Considering only complete Ab species (%)
** Considering all species (%)
*** Estimated change relative to wild type

*報告されたＫＤ値は、単回の測定または２回の測定の平均である。範囲は括弧内に示されている。 Table 31a: Biophysical characterization of selected designs derived from the D3H44 / trastuzumab system (antigen binding, thermal stability. UPLC-SEC)

* The reported KD value is the average of a single measurement or two measurements. The range is shown in parentheses.

*報告されたＫＤ値は、単回の測定または２回の測定の平均である。範囲は括弧内に示されている。 Table 31b: Biophysical characterization of selected designs derived from the D3H44 / cetuximab system (antigen binding, thermal stability. UPLC-SEC)

* The reported KD value is the average of a single measurement or two measurements. The range is shown in parentheses.

*報告されたＫＤ値は、単回の測定または２回の測定の平均である。範囲は括弧内に示されている。
**ＮＤ＝決定されず。 Table 31c: Biophysical characterization of selected designs derived from the trastuzumab / cetuximab system (antigen binding, thermal stability. UPLC-SEC)

* The reported KD value is the average of a single measurement or two measurements. The range is shown in parentheses.
** ND = not determined.

Table 32a. Effect of wild-type D3H44 / trastuzumab DNA titration ratio on the percentage of antibody species as assessed by LC-MS. H1 and L1 refer to the heavy and light chains of D3H44, respectively. H2 and L2 refer to the heavy and light chains of trastuzumab, respectively.

Table 32b. Effect of wild-type D3H44 / cetuximab DNA titration ratio on the percentage of antibody species as assessed by LC-MS. H1 and L1 refer to the heavy and light chains of D3H44, respectively. H2 and L2 refer to the heavy and light chains of cetuximab, respectively.

Table 32c: Effect of wild-type trastuzumab / cetuximab DNA titration ratio on the percentage of antibody species as assessed by LC-MS. H1 and L1 refer to the heavy and light chains of trastuzumab, respectively. H2 and L2 refer to the heavy and light chains of trastuzumab, respectively.

*完全なＡｂ種のみを考慮した（％）
**全ての種を考慮した（％） Table 33a: LC-MS pairing of wild type antibody constructs derived from the D3H44 (H1L1) / cetuximab (H2L2) bispecific system

* Considering only complete Ab species (%)
** Considering all species (%)

*完全なＡｂ種のみを考慮した（％）
**全ての種を考慮した（％） Table 33b LC-MS pairing of wild type antibody constructs derived from the D3H44 (H1L1) / trastuzumab (H2L2) bispecific system

* Considering only complete Ab species (%)
** Considering all species (%)

*完全なＡｂ種のみを考慮した（％）
**全ての種を考慮した（％） Table 33c LC-MS pairing of wild-type antibody constructs derived from the trastuzumab (H1L1) / cetuximab (H2L2) bispecific system

* Considering only complete Ab species (%)
** Considering all species (%)

*ＷＴは、野生型を指す
**ＮＡは、対応するＬＣＣＡセット番号がないので、該当なしを指す Table 34. Stabilization of mutations in Fab heterodimers.

* WT refers to wild type
** NA indicates not applicable since there is no corresponding LCCA set number

*完全なＡｂ種のみを考慮した（％）
**全ての種を考慮した（％）
***野生型に対する推定変化 Table 35a: Design showing mobility in both orientations in all three bispecific systems (D3H44 / cetuximab, D3H44 / trastuzumab, and trastuzumab / cetuximab)

* Considering only complete Ab species (%)
** Considering all species (%)
*** Estimated change relative to wild type

*完全なＡｂ種のみを考慮した（％）
**全ての種を考慮した（％）
***野生型に対する推定変化 (Table 35b) The mobility in all three bispecific systems (D3H44 / cetuximab, D3H44 / trastuzumab, and trastuzumab / cetuximab) is shown in one orientation and in the other orientation only in one bispecific system. Design that moves and meets at least 10% of light chain utilization criteria at the same time

* Considering only complete Ab species (%)
** Considering all species (%)
*** Estimated change relative to wild type

Claims (127)

An isolated antigen binding polypeptide construct comprising at least a first heterodimer and a second heterodimer comprising:
The first heterodimer comprises a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain polypeptide sequence (L1), and the second heterodimer comprises: A second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin light chain polypeptide sequence (L2), wherein at least one of the H1 or L1 sequences of the first heterodimer is Unlike the corresponding H2 or L2 sequence of the second heterodimer,
H1 and H2 each comprise at least a heavy chain variable domain (V _H domain) and a heavy chain constant domain (C _H1 domain);
Each of L1 and L2 comprises at least a light chain variable domain ( _VL domain) and a light chain constant domain ( _CL domain);
At least one of H1, H2, L1 and L2 contains at least one amino acid modification, wherein H1 preferentially pairs with L1 over L2, and H2 preferentially with L2 over L1 Or
H1, H2, L1 and L2 comprise a set of amino acid modifications, wherein H1 preferentially pairs with L1 over L2, H2 preferentially pairs with L2 over L1,
Corresponding that the thermal stability of the Fab region measured by melting temperature (Tm) of at least one of the first and second heterodimers does not have at least one amino acid modification or set of amino acid modifications An isolated antigen-binding polypeptide construct that is within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ° C. of the Tm of the heterodimer. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, K145, D146, Q179 and S186,
(Ii) The construct of claim 1, wherein L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at Q124, S131, V133, Q160, S176, T178 and T180. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124R, L124E, K145M, K145T, D146N, Q179E, Q179K, S186R and S186K;
(Ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q124E, S131R, S131K, V133G, Q160E, S176R, S176D, T178D, T178E and T180E. The construct described in 1. H1 comprises an amino acid modification selected from the group consisting of L124E, K145M, K145T and Q179E or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of S131R, S131K, V133G and S176R or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of L124R, D146N, Q179K, S186R and S186K, or combinations thereof;
4. The construct of claim 3, wherein L2 comprises an amino acid modification selected from the group consisting of Q124E, V133G, Q160E, S176D, T178D, T178E and T180E or combinations thereof. H1 includes amino acid modifications L124E, K145T and Q179E;
L1 comprises amino acid modifications S131K, V133G and S176R;
H2 includes amino acid modifications L124R and S186R;
The construct of claim 4, wherein L2 comprises the amino acid modifications V133G, S176D and T178D. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, L143, K145, D146, Q179 and S186;
The construct of claim 1, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at Q124, V133, Q160, S176, T178 and T180. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124E, L124R, L143E, L143D, K145T, K145M, D146N, Q179K, S186R and S186K;
(Ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q124K, Q124E, V133G, Q160K, S176R, S176D, T178E, T178K, T178R, T178D and T180E Item 7. The construct according to Item 6. H1 comprises an amino acid modification selected from the group consisting of L124E, L143E, L143D, K145T and K145M or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of Q124K, V133G, Q160K, S176R, T178K and T178R or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of L124R, D146N, Q179K, S186R and S186K, or combinations thereof;
8. The construct of claim 7, wherein L2 comprises an amino acid modification selected from the group consisting of Q124E, V133G, S176D, T178E, T178D and T180E or combinations thereof. H1 includes amino acid modifications L124E, L143E and K145T;
L1 comprises amino acid modifications Q124K, V133G and S176R;
H2 includes amino acid modifications L124R and Q179K;
9. The construct of claim 8, wherein L2 comprises amino acid modifications V133G, S176D, and T178E. H1 includes amino acid modifications L124E, L143E and K145T;
L1 comprises amino acid modifications Q124K, V133G and S176R;
H2 includes amino acid modifications L124R and S186R;
9. The construct of claim 8, wherein L2 comprises amino acid modifications V133G, S176D, and T178D. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications in Q39, L45, L124, L143, F122 and H172;
(Ii) The construct of claim 1, wherein L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at Q38, P44, Q124, S131, V133, N137, S174, S176 and T178. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from Q39E, Q39R, L45P, F122C, L124E, L124R, L143F, H172T and H172R;
(Ii) L1 and / or L2 is at least one amino acid modification or amino acid modification selected from Q38R, Q38E, P44F, Q124C, S131T, S131E, V133G, N137K, S174R, S176R, S176K, S176D, T178Y and T178D 12. The construct of claim 11, comprising a set of: H1 comprises an amino acid modification selected from the group consisting of Q39E, L45P, F122C, L124E, L143F, H172T and H172R or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of Q38R, P44F, Q124C, S131T, V133G, N137K, S174R, S176R, S176K and T178Y, or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of Q39R, L124R and H172R or combinations thereof;
13. The construct of claim 12, wherein L2 comprises an amino acid modification selected from the group consisting of Q38E, S131E, V133G, S176D and T178D or combinations thereof. H1 includes amino acid modifications Q39E and L124E;
L1 comprises amino acid modifications Q38R, V133G and S176R;
H2 includes amino acid modifications Q39R and L124R;
14. The construct of claim 13, wherein L2 comprises amino acid modifications Q38E, V133G and S176D. H1 includes amino acid modifications L45P and L124E;
L1 includes amino acid modifications P44F, V133G and S176R;
H2 contains the amino acid modification L124R;
14. The construct of claim 13, wherein L2 comprises amino acid modifications V133G, S176D, and T178D. H1 includes amino acid modifications L124E and L143F;
L1 includes amino acid modifications V133G and S176R;
H2 contains the amino acid modification L124R;
14. The construct of claim 13, wherein L2 comprises amino acid modifications V133G, S176D, and T178D. H1 includes amino acid modifications F122C and L124E;
L1 comprises amino acid modifications Q124C, V133G and S176R,
H2 contains the amino acid modification L124R;
14. The construct of claim 13, wherein L2 comprises amino acid modifications V133G and S176D. H1 includes amino acid modifications L124E and H172T;
L1 comprises amino acid modifications V133G, N137K, S174R and S176R,
H2 includes amino acid modifications L124R and H172R;
14. The construct of claim 13, wherein L2 comprises amino acid modifications V133G, S176D, and T178D. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, A125, H172 and K228;
The construct of claim 1, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at S121, V133, N137, S174, S176 and T178. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124E, L124R, A125S, A125R, H172R, H172T and K228D;
20. The construct of claim 19, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from S121K, V133G, N137K, S174R, S176K, S176R, S176D and T178D. . H1 comprises an amino acid modification selected from the group consisting of L124E, A125S, H172R and K228D or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of S121K, V133G and S176R or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of L124R, A125R and H172T or combinations thereof;
21. The construct of claim 20, wherein L2 comprises an amino acid modification selected from the group consisting of V133G, N137K, S174R, S176D and T178D or combinations thereof. H1 comprises amino acid modifications L124E and K228D,
L1 includes amino acid modifications S121K, V133G and S176R;
H2 includes amino acid modifications L124R and A125R;
24. The construct of claim 21, wherein L2 comprises amino acid modifications V133G and S176D. H1 includes amino acid modifications L124E and H172R;
L1 includes amino acid modifications V133G and S176R;
H2 includes amino acid modifications L124R and H172T;
23. The construct of claim 21, wherein L2 comprises amino acid modifications V133G, S174R, and S176D. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, A139 and V190;
The construct of claim 1, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at F116, V133, L135 and S176. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124E, L124R, A139W, A139G and V190A;
25. The construct of claim 24, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from F116A, V133G, L135V, L135W, S176R and S176D. H1 comprises an amino acid modification selected from the group consisting of L124E and A139W or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of F116A, V133G, L135V and S176R or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of L124R, A139G and V190A or combinations thereof;
26. The construct of claim 25, wherein L2 comprises an amino acid modification selected from the group consisting of V133G, L135W and S176D or combinations thereof. H1 includes amino acid modifications L124E and A139W;
L1 includes amino acid modifications F116A, V133G, L135V and S176R;
H2 includes amino acid modifications L124R, A139G and V190A;
27. The construct of claim 26, wherein L2 comprises amino acid modifications V133G, L135W and S176D. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at Q39, L45, K145, H172, Q179 and S186,
(Ii) The construct according to claim 1, wherein L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications in Q38, P44, Q124, S131, Q160, T180 and C214. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from Q39E, Q39R, L45P, K145T, H172R, Q179E and S186R;
29. The construct of claim 28, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q38R, Q38E, P44F, Q124E, S131K, Q160E, T180E and C214S. . H1 comprises an amino acid modification selected from the group consisting of Q39E, L45P, K145T, H172R and Q179E or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of Q38R, P44F and S131K or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of Q39R, H172R and S186R or combinations thereof;
30. The construct of claim 29, wherein L2 comprises an amino acid modification selected from the group consisting of Q38E, Q124E, Q160E, T180E and C214S or combinations thereof. H1 comprises amino acid modifications Q39E, K145T and Q179E,
L1 includes amino acid modifications Q38R and S131K;
H2 includes amino acid modifications Q39R and S186R;
31. The construct of claim 30, wherein L2 comprises amino acid modifications Q38E, Q124E, Q160E and T180E. H1 includes amino acid modifications L45P, K145T, H172R and Q179E;
L1 includes amino acid modifications P44F and S131K;
H2 includes amino acid modifications H172R and S186R;
32. The construct of claim 30, wherein L2 comprises amino acid modifications Q124E, Q160E and T180E. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at A139, L143, K145, Q179 and V190;
(Ii) The construct of claim 1, wherein L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at F116, Q124, L135, Q160, T178 and T180. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from A139W, A139G, L143E, K145T, Q179E, Q179K and V190A;
34. The construct of claim 33, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from F116A, Q124R, Q124E, L135V, L135W, Q160E, T178R and T180E. . H1 comprises an amino acid modification selected from the group consisting of A139W, L143E, K145T and Q179E or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of F116A, Q124R, L135V and T178R or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of A139G, Q179K and V190A or combinations thereof;
35. The construct of claim 34, wherein L2 comprises an amino acid modification selected from the group consisting of Q124E, L135W, Q160E and T180E or combinations thereof. H1 comprises amino acid modifications A139W, L143E, K145T and Q179E,
L1 includes amino acid modifications F116A, Q124R, L135V and T178R;
H2 contains the amino acid modification Q179K;
36. The construct of claim 35, wherein L2 comprises amino acid modifications Q124E, L135W, Q160E and T180E. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications in Q39, L143, K145, D146, H172 and Q179;
(Ii) The construct of claim 1, wherein L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at Q38, Q124, Q160, T178 and T180. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from Q39E, Q39R, L143E, K145T, D146G, H172R, Q179E and Q179K;
38. The construct of claim 37, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q38R, Q38E, Q124R, Q124E, Q160K, Q160E, T178R and T180E. . H1 comprises an amino acid modification selected from the group consisting of Q39E, L143E, K145T, H172R and Q179E or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of Q38R, Q124R, Q160K and T178R or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of Q39R, H172R and Q179K or combinations thereof;
40. The construct of claim 38, wherein L2 comprises an amino acid modification selected from the group consisting of Q38E, Q124E, D146G, Q160E and T180E or combinations thereof. H1 comprises amino acid modifications Q39E, L143E, K145T and Q179E,
L1 comprises amino acid modifications Q38R, Q124R, Q160K and T178R;
H2 comprises amino acid modifications Q39R, H172R and Q179K,
40. The construct of claim 39, wherein L2 comprises amino acid modifications Q38E, Q124E, Q160E, and T180E. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L45, L143, K145, D146, H172 and Q179;
(Ii) The construct according to claim 1, wherein L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at Q38, P44, Q124, N137, Q160, S174, T178, T180 and C214. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L45P, L143E, K145T, D146G, H172R, H172T, Q179E and Q179K;
(Ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q38E, P44F, Q124R, Q124E, N137K, Q160K, Q160E, S174R, T178R, T180E and C214S Item 42. The construct according to Item 41. H1 comprises an amino acid modification selected from the group consisting of L45P, L143E, K145T, H172R and Q179E or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of P44F, Q124R, Q160K and T178R or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of D146G, H172R, H172T and Q179K or combinations thereof;
43. The construct of claim 42, wherein L2 comprises an amino acid modification selected from the group consisting of Q38E, Q124E, N137K, Q160E, S174R, T180E and C214S or combinations thereof. H1 comprises amino acid modifications L45P, L143E and K145T;
L1 includes amino acid modifications P44F, Q124R, Q160K and T178R;
H2 comprises amino acid modifications D146G and Q179K,
44. The construct of claim 43, wherein L2 comprises amino acid modifications Q38E, Q124E, Q160E, and T180E. H1 comprises amino acid modifications L143E, K145T and H172R;
L1 includes amino acid modifications Q124R, Q160K and T178R;
H2 comprises the amino acid modifications H172T and Q179K,
44. The construct of claim 43, wherein L2 comprises amino acid modifications Q124E, Q160E, N137K, S174R and T180E. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, L143, K145 and Q179;
(Ii) The construct according to claim 1, wherein L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at Q124, S131, V133, S176, T178 and T180. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from L124W, L124A, L143E, L143F, K145T, Q179E and Q179K;
(Ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from Q124R, Q124K, Q124E, S131K, V133A, V133W, S176T, T178R, T178L, T178E and T180E Item 47. The construct according to Item 46. H1 comprises an amino acid modification selected from the group consisting of L124W, L143E, K145T and Q179E or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of Q124R, Q124K, S131K, V133A, S176T, T178R and T178L or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of L124A, L143F and Q179K or combinations thereof;
48. The construct of claim 47, wherein L2 comprises an amino acid modification selected from the group consisting of Q124E, V133W, S176T, T178L, T178E and T180E or combinations thereof. H1 includes amino acid modifications L124W, L143E, K145T and Q179E;
L1 comprises amino acid modifications Q124R, V133A, S176T and T178R;
H2 includes amino acid modifications L124A, L143F and Q179K;
49. The construct of claim 48, wherein L2 comprises amino acid modifications Q124E, V133W, S176T, T178L and T180E. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at A139, L143, K145, Q179 and S186,
The construct of claim 1, wherein (ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at F116, Q124, V133, Q160, T178 and T180. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications selected from A139C, L143E, L143D, L143R, L143K, K145T, Q179E, Q179D, Q179R, Q179K, S186K, S186R ,
(Ii) L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications selected from F116C, Q124R, Q124K, Q124E, V133E, V133D, Q160K, Q160E, T178R, T178K, T178E and T180E 51. The construct of claim 50. H1 comprises an amino acid modification selected from the group consisting of A139C, L143E, L143D, K145T, Q179E and Q179D or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of F116C, Q124R, Q124K, Q160K, T178R and T178K or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of L143R, L143K, Q179R, Q179K, S186K and S186R or combinations thereof;
52. The construct of claim 51, wherein L2 comprises an amino acid modification selected from the group consisting of Q124E, V133E, V133D, Q160E, T178E and T180E or combinations thereof. H1 comprises amino acid modifications A139C, L143E, K145T and Q179E,
L1 comprises amino acid modifications F116C, Q124R and T178R;
H2 contains the amino acid modification Q179K;
53. The construct of claim 52, wherein L2 comprises amino acid modifications Q124E, Q160E, and T180E. H1 comprises amino acid modifications L143E, K145T and Q179E,
L1 includes amino acid modifications Q124R and T178R;
H2 contains the amino acid modification S186K,
53. The construct of claim 52, wherein L2 comprises amino acid modifications Q124E, Q160E and T178E. H1 comprises amino acid modifications L143E, K145T and Q179E,
L1 includes amino acid modifications Q124R and T178R;
H2 contains the amino acid modification L143R;
53. The construct of claim 52, wherein L2 comprises amino acid modifications Q124E and V133E. (I) H1 and / or H2 comprises at least one amino acid modification or set of amino acid modifications at L124, L143, K145, D146, Q179, S186 and S188,
(Ii) The construct of claim 1, wherein L1 and / or L2 comprises at least one amino acid modification or set of amino acid modifications at Q124, S131, V133, Q160, S176, T178 and T180. (I) at least one amino acid modification or set of amino acid modifications wherein H1 and / or H2 is selected from L124A, L143A, L143R, L143E, L143K, K145T, D146G, Q179R, Q179E, Q179K, S186R, S186K and S188L Including
(Ii) L1 and / or L2 is at least one selected from Q124R, Q124E, S131E, S131T, V133Y, V133W, V133E, V133D, Q160E, Q160K, Q160M, S176L, T178R, T178E, T178F, T178Y and T180E 57. The construct of claim 56, comprising an amino acid modification or a set of amino acid modifications. H1 comprises an amino acid modification selected from the group consisting of L143E, K145T, Q179E and S188L or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of Q124R, Q160K and T178R or combinations thereof;
H2 comprises an amino acid modification selected from the group consisting of L124A, L143A, L143R, L143K, D146G, Q179R, Q179K, S186R and S186K, or combinations thereof;
58. In claim 57, L2 comprises an amino acid modification selected from the group consisting of Q124E, S131E, S131T, V133Y, V133W, V133E, V133D, Q160E, Q160M, S176L, T178E, T178F, T178Y and T180E or combinations thereof. The described construction. H1 comprises amino acid modifications L143E, K145T, Q179E and S188L;
L1 includes amino acid modifications Q124R and T178R;
H2 contains the amino acid modification S186K,
59. The construct of claim 58, wherein L2 comprises amino acid modifications Q124E, S176L and T180E. H1 comprises amino acid modifications L143E, K145T, Q179E and S188L;
L1 includes amino acid modifications Q124R and T178R;
H2 contains the amino acid modification S186K,
59. The construct of claim 58, wherein L2 comprises amino acid modifications Q124E, S131T, T178Y and T180E. H1 includes amino acid modifications L143E and K145T;
L1 includes amino acid modifications Q124R, Q160K and T178R;
H2 contains the amino acid modification S186K,
59. The construct of claim 58, wherein L2 comprises amino acid modification S131E. H1 includes amino acid modifications L143E and K145T;
L1 contains the amino acid modification Q124R;
H2 contains the amino acid modification L143R;
59. The construct of claim 58, wherein L2 comprises amino acid modifications Q124E and V133E. (I) H1 comprises at least one amino acid modification or set of amino acid modifications at F122 and C233;
(Ii) The construct of claim 1, wherein L1 comprises at least one amino acid modification or set of amino acid modifications at Q124 and C214. (I) H1 comprises at least one amino acid modification or set of amino acid modifications selected from F122C and C233S;
64. The construct of claim 63, wherein (ii) L1 comprises at least one amino acid modification or set of amino acid modifications selected from Q124C and C214S. H1 comprises an amino acid modification selected from the group consisting of F122C and C233S or combinations thereof;
L1 comprises an amino acid modification selected from the group consisting of Q124C and C214S or a combination thereof;
H2 comprises a wild type or unmodified amino acid sequence;
65. The construct of claim 64, wherein L2 comprises a wild type or unmodified amino acid sequence. H1 includes amino acid modifications F122C and C233S;
L1 includes amino acid modifications Q124C and C214S;
H2 comprises a wild type or unmodified amino acid sequence;
66. The construct of claim 65, wherein L2 comprises a wild type or unmodified amino acid sequence.   When H1, H2, L1 and L2 are coexpressed in cells or mammalian cells; or when H1, H2, L1 and L2 are coexpressed in a cell-free expression system; or H1, H2, L1 and L2 are co-produced Or when H1, H2, L1 and L2 are co-produced via a redox production method, H1 preferentially paired with L1 over L2, and H2 preferentially paired with L2 over L1. A construct according to any one of the preceding claims.   At least one of H1, H2, L1 and L2 is VH and H1 is preferentially paired with L1 over L2 and / or H2 is preferentially paired with L2 over L1. The construct according to any one of the preceding claims, comprising at least one amino acid modification of the VL domain and / or at least one amino acid modification of the CH1 and / or CL domains.   When H1 contains at least one amino acid modification in the CH1 domain, at least one of L1 and L2 contains at least one amino acid modification in the CL domain; and / or H1 is at least in the VH domain If it contains one amino acid modification, at least one of L1 and L2 contains at least one amino acid modification in the VL domain; or if H2 contains at least one amino acid modification in the CH1 domain, And at least one of L2 comprises at least one amino acid modification in the CL domain; and / or when H2 comprises at least one amino acid modification in the VH domain, at least one of L1 and L2 is The VL domain contains at least one amino acid modification Construct according to any one of the preceding claims.   Any one of the preceding claims, wherein H1, L1, H2 and / or L2 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations in the Fab region. The construct according to item.   At least one of H1, H2, L1 and L2 has at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications in at least one constant domain and / or at least one variable domain. A construct according to any one of the preceding claims comprising.   When both L1 and L2 are co-expressed with at least one of H1 and H2, the relative pairing of at least one of the H1-L1 and H2-L2 heterodimer pairs is the corresponding H1-L2 respectively. Or for H2-L1 heterodimer pairs, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 94, 95, 96, 97, 98 or more than 99%, and the relative pairing of the modified H1-L1 or H2-L2 heterodimer pair does not have at least one amino acid modification H1-L1 or H2-L Greater than their relative pairing observed in heterodimer pairs, construct according to any one of the preceding claims.   The thermal stability of the Fab region in at least one of the first and second heterodimers is such that the Tm of the corresponding heterodimer without at least one amino acid modification or set of amino acid modifications. The construct according to any one of the preceding claims, which is within about 0, 1, 2 or 3 ° C.   The affinity of each heterodimer for the antigen to which it binds is about 1, 2 of the affinity of the corresponding unmodified heterodimer for the same antigen as measured by surface plasmon resonance (SPR) or FACS. The construct according to any one of the preceding claims, which is within 3, 3, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45 or 50 times. When H1 is paired with L1 rather than L2, at least one of H1 and L1 comprises at least one domain comprising at least one amino acid modification that results in greater steric complementarity of the amino acids, or H2 When paired with L2 rather than L1, at least one of H2 and L2 contains at least one domain comprising at least one amino acid modification that results in greater steric complementarity of the amino acids, or H1 is more than L2 When paired with L1, at least one of H1 and L1 contains at least one domain comprising at least one amino acid modification that results in greater electrostatic complementarity between charged amino acids, or H2 is more than L1 When paired with L2, at least one of H2 and L2 is greater between charged amino acids. Or at least one domain containing at least one amino acid modification that results in electrostatic complementarity, or when H1 pairs with L1 rather than L2, at least one of H1 and L1 has a larger conformation of amino acids. And at least one domain comprising at least one amino acid modification that results in electrostatic complementarity, or when H2 pairs with L2 rather than L1, at least one of H2 and L2 has a larger conformation of amino acids. And at least one domain comprising at least one amino acid modification that results in electrostatic complementarity, or at least one of H1 and L1 comprises at least one amino acid modification that results in a covalent bond between H1 and L1. Contains at least one domain, or at least one of H2 and L2 is H When at least one domain comprising at least one amino acid modification results in a covalent bond between the L2, construct according to any one of the preceding claims.   Wherein H1, H2, L1 and L2 comprise a set of amino acid modifications selected from the group consisting of the set of unique identifier designs shown in Tables 5-6, 15-27 or 28a-28c. The construct according to any one of the above.   The construct further comprises an Fc comprising a first CH3 sequence and a second CH3 sequence, wherein the first CH3 sequence is the first heterogeneous with or without one or more linkers. 8. The method of any one of the preceding claims, wherein the second CH3 sequence binds to the second heterodimer with or without one or more linkers. The construct described in 1.   78. The Fc is human Fc, human IgG1 Fc, human IgA Fc, human IgG Fc, human IgD Fc, human IgE Fc, human IgM Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc. Constructs.   79. The construct of claim 77 or 78, wherein the Fc is a heterodimeric Fc.   80. The construct according to any one of claims 77 to 79, wherein the Fc comprises one or more modifications in at least one of the CH3 sequences.   The dimerized CH3 sequence is approximately 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, 83, as measured by DSF. 81. A construct according to any one of claims 77-80 having a melting temperature (Tm) of 84 or 85 [deg.] C or higher.   When the Fc is produced, it is about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, A heterodimer formed with a purity greater than 95, 96, 97, 98 or 99%, or when the Fc is expressed or expressed via a single cell, about 75, 76, 77 , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% purity or more 82. The construct according to any one of claims 77 to 81, wherein the construct is a heterodimer that is produced.   78. The Fc comprises one or more modifications in at least one of the CH3 sequences that promote the formation of a heterodimeric Fc with stability comparable to a wild type homodimeric Fc. 83. A construct according to any one of -82. The Fc is
i) a heterodimeric IgG1 Fc having a modified L351Y_F405A_Y407V in the first Fc polypeptide and a modified T366L_K392M_T394W in the second Fc polypeptide;
ii) a heterodimeric IgG1 Fc having a modified L351Y_F405A_Y407V in the first Fc polypeptide and a modified T366L_K392L_T394W in the second Fc polypeptide;
iii) a heterodimeric IgG1 Fc having a modified T350V_L351Y_F405A_Y407V in the first Fc polypeptide and a modified T350V_T366L_K392L_T394W in the second Fc polypeptide;
iv) a heterodimeric IgG1 Fc having a modified T350V_L351Y_F405A_Y407V in the first Fc polypeptide and a modified T350V_T366L_K392M_T394W in the second Fc polypeptide; or v) having a modified T350V_L351Y_S400E_F40 Heterodimeric IgG1 Fc with modified T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide
85. A construct according to any one of claims 78 to 84, comprising:   85. The construct according to any one of claims 77 to 84, wherein the Fc further comprises at least one CH2 sequence.   86. The construct of claim 85, wherein the CH2 sequence of the Fc comprises one or more modifications.   87. The construct of any one of claims 77-86, wherein the Fc comprises one or more modifications that promote selective binding of Fc gamma receptors.   88. The method of any one of claims 77-87, wherein the Fc is bound to the heterodimer by one or more linkers, or the Fc is bound to H1 and H2 by one or more linkers. The described construction.   90. The construct of claim 88, wherein the one or more linkers are one or more polypeptide linkers.   90. The construct of claim 88, wherein the one or more linkers comprise one or more antibody hinge regions.   90. The construct of claim 88, wherein the one or more linkers comprise one or more IgGl hinge regions.   92. The construct according to any one of claims 88 to 91, wherein the one or more linkers comprise one or more modifications.   94. The construct of claim 92, wherein the one or more modifications promote selective binding of an Fc gamma receptor.   The construct according to any one of the preceding claims, wherein the at least one amino acid modification is at least one amino acid mutation, or the at least one amino acid modification is at least one amino acid substitution. .   The construct according to any one of the preceding claims, wherein each sequence of H1, H2, L1 and L2 is derived from a human sequence.   A construct according to any one of the preceding claims, which is multispecific or bispecific.   A construct according to any one of the preceding claims, which is multivalent or divalent.   An isolated polynucleotide or set of isolated polynucleotides comprising at least one sequence encoding a construct according to any one of the preceding claims.   99. The isolated polynucleotide or set of isolated polynucleotides of claim 98, wherein the polynucleotide or set of polynucleotides is cDNA.   100. A vector or set of vectors comprising one or more of the polynucleotides of claim 98 or 99 or a set of polynucleotides.   101. The vector or vector set of claim 100, selected from the group consisting of a plasmid, a multicistronic vector, a viral vector, a non-episomal mammalian vector, an expression vector and a recombinant expression vector.   101. An isolated cell comprising the polynucleotide or set of polynucleotides of claim 98 or 99, or the vector or set of vectors of claim 100 or 101.   103. The isolated cell of claim 102, which is a yeast cell, bacterial cell, hybridoma, Chinese hamster ovary (CHO) cell or HEK293 cell.   A pharmaceutical composition comprising the construct according to any one of the preceding claims and a pharmaceutically acceptable carrier.   105. Further comprising one or more substances selected from the group consisting of buffers, antioxidants, low molecular weight molecules, drugs, proteins, amino acids, carbohydrates, lipids, chelating agents, stabilizers and excipients. A pharmaceutical composition according to 1.   99. Use of a construct according to any one of claims 1 to 96 or a pharmaceutical composition according to claim 104 or 105 for the treatment of a disease or disorder in a subject or in the manufacture of a medicament.   A method of treating a subject having a disease or disorder comprising the step of administering to the subject a construct according to any one of claims 1 to 96 or a pharmaceutical composition according to claim 104 or 105. Said method. A method of obtaining a construct according to any one of claims 1 to 96 from a host cell culture comprising:
(A) obtaining a host cell culture comprising at least one host cell comprising one or more nucleic acid sequences encoding said construct;
(B) recovering the construct from the host cell culture. A method of obtaining a construct according to any one of claims 1 to 96, comprising:
(A) obtaining H1, L1, H2 and L2;
(B) preferentially pairing H1 with L1 over L2, and preferentially pairing H2 with L2 over L1;
(C) obtaining the construct. 99. A method of preparing a construct according to any one of claims 1 to 96, comprising the following steps:
a. Obtaining a polynucleotide or set of polynucleotides encoding at least one construct;
b. Determining an optimal ratio of each of the polynucleotide or set of polynucleotides for introduction into at least one host cell, wherein the optimal ratio is determined upon expression of H1, L1, H2, and L2. H1-L1 and H2-L2 heterodimer pairs formed upon expression of H1, L1, H2 and L2 compared to the mismatched H1-L2 and H2-L1 heterodimer pairs formed A stage determined by assessing the amount of;
c. Selecting a preferred optimal ratio, wherein transfection of at least one host cell with said preferred optimal ratio of said polynucleotide or set of polynucleotides results in expression of said construct;
d. Transfecting said at least one host cell with said optimal ratio of said polynucleotide or set of polynucleotides;
e. Culturing the at least one host cell to express the construct.   111. The method of claim 110, wherein selecting the optimal ratio is assessed by transfection in a transient transfection system.   112. The method of claim 110 or 111, wherein transfection of the at least one host cell with the preferred optimal ratio of the polynucleotide or set of polynucleotides results in optimal expression of the construct.   The construct comprises an Fc comprising at least two CH3 sequences, wherein the Fc is with or without one or more linkers, the first heterodimer and the second heterodimer. 113. A method according to any one of claims 110 to 112, wherein the method binds to a body.   114. The method of claim 113, wherein the Fc is a heterodimer optionally comprising one or more amino acid modifications. A first heterodimer comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain polypeptide sequence (L1); and a second immunoglobulin heavy chain polypeptide sequence ( A data set comprising data representing a complementary mutation in a second heterodimer comprising H2) and a second immunoglobulin light chain polypeptide sequence (L2) comprising:
H1 and H2 each comprise at least a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain);
L1 and L2 each comprise at least a light chain variable domain (VL domain) and a light chain constant domain (CL domain);
A computer readable storing said data set, wherein the data set of complementary mutations comprises data representing the mutations presented in Tables 4-6, 15-27, 28a-28c or a subset of these mutations Possible storage medium. A method of producing a bispecific antigen binding polypeptide construct, wherein the bispecific construct comprises:
A first heterodimer comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain polypeptide sequence (L1) from a first monospecific antigen binding polypeptide A second heteroduplex comprising a second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin light chain polypeptide sequence (L2) from a second monospecific antigen binding polypeptide; Including masses,
H1 and H2 each comprise at least a heavy chain variable domain (VH domain) and a heavy chain constant domain (CH1 domain);
L1 and L2 each comprise at least a light chain variable domain (VL domain) and a light chain constant domain (CL domain);
a. Introducing one or more complementary mutations from the data set of claim 115 into the first heterodimer and / or the second heterodimer;
b. Co-expressing the first heterodimer and the second heterodimer in at least one host cell to produce an expression product comprising the bispecific construct.   117. The method of claim 116, further comprising determining the amount of the bispecific construct in the expression product relative to other polypeptide products to select a preferred subset of complementary mutations.   118. The method of claim 116 or 117, wherein the bispecific construct is produced with a purity of greater than 70% compared to the other polypeptide product.   117.Adding additional amino acid modifications to at least one of H1, H2, L1 or L2 to further increase the purity of the bispecific construct relative to the other polypeptide product. 119. The method according to any one of -118.   The construct comprises an Fc comprising at least two CH3 sequences, wherein the Fc is with or without one or more linkers, the first heterodimer and the second heterodimer. 120. A method according to any one of claims 116 to 119, wherein the method is associated with a body.   121. The method of claim 120, wherein said Fc is a heterodimer optionally comprising one or more amino acid modifications.   122. The method according to any one of claims 116 to 121, wherein the antigen-binding polypeptide is an antibody, a single chain monoclonal antibody, a Fab or a single chain Fab. A method of preparing an isolated antigen binding polypeptide construct according to any one of claims 1 to 96, comprising:
Expressing H1, H2, L1 and L2 in at least one expression system to produce an expression product;
Characterizing the expression product; and selecting the expression system in which more than 90% of the expression product is the isolated antigen-binding polypeptide construct. Said amino acid modification is a unique identifier of Table 35a:
H1 includes L124W, L143E, K145T and Q179E, L1 includes Q124R, V133A, S176T and T178R, H2 includes L124A, L143F and Q179K, L2 includes Q124E, V133W, S176T, T178L and T180E, 9561- 9095_1, 9561-9095_2;
H1 includes L124E and H172T, L1 includes V133G, N137K, S174R and S176R, H2 includes L124R and H172R, L2 includes V133G, S176D and T178D, 9121-9373_1, 9121-9373_2;
H1 includes L124E and A139W, L1 includes F116A, V133G, L135V and S176R, H2 includes L124R, A139G and VI90A, L2 includes V133G, L135W and S176D, 9116-9349_1, 9116-9349_2;
H1 includes L124E, K145T and Q179E, L1 includes S131K, V133G and S176R, H2 includes L124R and S186R, L2 includes V133G, S176D and T178D, 9134-9521_1, 9134-9521_2;
H1 includes L124E, L143E and K145T, L1 includes Q124K, V133G and S176R, H2 includes L124R and Q179K, L2 includes V133G, S176D and T178E, 9286-9402_1, 9286-9402_2;
H1 includes L143E, K145T and Q179E, L1 includes Q124R and T178R, H2 includes S186K, L2 includes Q124E, Q160E and T178E, 9667-9830_1, 9667-9830_2;
H1 includes L143E, K145T, Q179E, and S188L, L1 includes Q124R and T178R, H2 includes S186K, L2 includes Q124E, S176L, and T180E, 9696-9848_1, 9696-9848_2;
H1 includes A139W, L143E, K145T and Q179E, L1 includes F116A, Q124R, L135V and T178R, H2 includes Q179K, L2 includes Q124E, L135W, Q160E and T180E, 9060-9756_1, 9060-9756_2;
H1 includes L143E, K145T and Q179E, L1 includes Q124R and T178R, H2 includes L143R, L2 includes Q124E and V133E, 9682-9740_1, 9682-9740_2;
H1 includes A139C, L143E, K145T and Q179E, L1 includes F116C, Q124R and T178R, H2 includes Q179K, L2 includes Q124E, Q160E and T180E, 9049-9759_1, 9049-9759_2; and H1 includes Q39E , L143E, K145T and Q179E, L1 includes Q38R, Q124R, Q160K and T178R, H2 includes Q39R, H172R and Q179K, L2 includes Q38E, Q124E, Q160E and T180E, 9820-9823_1, 9820-9823_2
The construct of claim 1, selected from SMCA designs corresponding to: Said amino acid modification is a unique identifier of Table 35b:
H1 includes L124E and L143F, L1 includes V133G and S176R, H2 includes L124R, L2 includes V133G, S176D and T178D, 9327-6054_1;
H1 includes Q39E and L124E, L1 includes Q38R, V133G and S176R, H2 includes Q39R and L124R, L2 includes Q38E, V133G and S176D, 9815-9825_1, 9815-9825_2;
H1 includes L143E and K145T, L1 includes Q124R, H2 includes L143R, L2 includes Q124E and V133E, 9587-9735_1, 9587-9735_2;
H1 includes L45P, L143E and K145T, L1 includes P44F, Q124R, Q160K and T178R, H2 includes D146G and Q179K, L2 includes Q124E, Q160E and T180E, 3522_1, 3522_2; and H1 includes L45P, K145T , H172R and Q179E, L1 includes P44F and S131K, H2 includes H172R and S186R, L2 includes Q124E, Q160E and T180E, 3519_1, 3519_2
The construct of claim 1, selected from SMCA designs corresponding to: Said amino acid modification is a unique identifier:
H1 includes A139W, L143E, K145T and Q179E, L1 includes F116A, Q124R, L135V and T178R, H2 includes Q179K, L2 includes Q124E, L135W, Q160E and T180E, 9060-9756;
H1 includes Q39E, L143E, K145T and Q179E, L1 includes Q38R, Q124R, Q160K and T178R, H2 includes Q39R, H172R and Q179K, L2 includes Q38E, Q124E, Q160E and T180E; 9820-9823;
H1 includes L45P, K145T, H172R and Q179E, L1 includes P44F and S131K, H2 includes H172R and S186R, L2 includes Q124E, Q160E and T180E, 3519;
H1 includes A139C, L143E, K145T and Q179E, L1 includes F116C, Q124R and T178R, H2 includes Q179K, L2 includes Q124E, Q160E and T180E, 9049-9759;
H1 includes L45P, L143E and K145T, L1 includes P44F, Q124R, Q160K and T178R, H2 includes D146G and Q179K, L2 includes Q124E, Q160E and T180E, 3522;
H1 includes L143E, K145T, Q179E and S188L, L1 includes Q124R and T178R, H2 includes S186K, L2 includes Q124E, S176L and T180E, 9696-9848;
H1 includes L143E, K145T, Q179E and S188L, L1 includes Q124R and T178R, H2 includes S186K, L2 includes Q124E, S131T, T178Y and T180E, 9692-9646;
H1 includes L143E and K145T, L1 includes Q124R, Q160K and T178R, H2 includes S186K, L2 includes S131E, 9986-9978; and H1 includes L143E, K145T and Q179E, and L1 includes Q124R and T178R H2 includes S186K, L2 includes Q124E, Q160E, and T178E, 9667-9830
The construct of claim 1, selected from SMCA designs corresponding to: Said amino acid modification is a unique identifier:
H87 includes L143E and K145T, L1 includes Q124R, H2 includes L143R, and L2 includes Q124E and V133E, 9587-9735;
H1 includes L124W, L143E, K145T and Q179E, L1 includes Q124R, V133A, S176T and T178R, H2 includes L124A, L143F and Q179K, L2 includes Q124E, V133W, S176T, T178L and T180E, 9561- 9095;
H1 includes L143E, K145T and H172R; L1 includes Q124R, Q160K and T178R; H2 includes H172T and Q179K; L2 includes Q124E, N137K, Q160E, S174R and T180E; 9611-9077;
H1 includes L124E and K228D, L1 includes S121K, V133G and S176R, H2 includes L124R and A125R, L2 includes V133G and S176D, 9168-9342;
H1 includes L124E, K145T and Q179E, L1 includes S131R, V133G and S176R, H2 includes L124R and S186R, L2 includes V133G, S176D and T180E, 9164-9555;
H1 includes L124E, L143E and K145T, L1 includes Q124K, V133G and S176R, H2 includes L124R and S186R, L2 includes V133G, S176D and T178D, 9279-9518;
H1 includes L124E, L143E and K145T, L1 includes Q124K, V133G and S176R, H2 includes L124R and Q179K, L2 includes V133G, S176D and T180E, 9290-9432;
H1 includes L124E, K145T and Q179E, L1 includes S131K, V133G and S176R, H2 includes L124R and Q179K, L2 includes V133G, S176D and T178E, 9142-9414;
H1 includes A139W, L143E, K145T and Q179E, L1 includes F116A, Q124R, L135V and T178R, H2 includes Q179K, L2 includes Q124E, L135W, Q160E and T180E, 9060-9756;
H1 includes L124E and H172T, L1 includes V133G, N137K, S174R and S176R, H2 includes L124R and H172R, L2 includes V133G, S176D and T178D, 9121-9373;
H1 includes F122C and L124E, L1 includes Q124C, V133G and S176R, H2 includes L124R, L2 includes V133G and S176D, 9066-9335;
H1 includes Q39E, L143E, K145T and Q179E, L1 includes Q38R, Q124R, Q160K and T178R, H2 includes Q39R, H172R and Q179K, L2 includes Q38E, Q124E, Q160E and T180E; 9820-9823;
H1 includes Q39E, K145T and Q179E, L1 includes Q38R and S131K, H2 includes Q39R and S186R, L2 includes Q38E, Q124E, Q160E and T180E, 9814-9828;
H1 includes L143E, K145T, Q179E and S188L, L1 includes Q124R and T178R, H2 includes S186K, L2 includes Q124E, S176L and T180E, 9696-9848;
H1 includes L143E, K145T and Q179E, L1 includes Q124R and T178R, H2 includes S186K, L2 includes Q124E, Q160E and T178E, 9667-9830;
9986-9978, where H1 includes L143E and K145T, L1 includes Q124R, Q160K, and T178R, H2 includes S186K, and L2 includes S131E;
H1 includes L45P, L143E and K145T, L1 includes P44F, Q124R, Q160K and T178R, H2 includes D146G and Q179K, L2 includes Q124E, Q160E and T180E, 3522; and H1 includes L45P, K145T and H172R And Q179E, L1 includes P44F and S131K, H2 includes H172R and S186R, and L2 includes Q124E, Q160E, and T180E, 3519
The construct of claim 1, selected from SMCA designs corresponding to:

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