KR20230019503A - Modified antigen binding polypeptide constructs and uses thereof - Google Patents

Abstract

Provided herein are antigen binding polypeptide constructs that may include a first heterodimer and a second heterodimer, each heterodimer comprising an immunoglobulin heavy chain or fragment thereof and an immunoglobulin light chain or fragment thereof. At least one of the heterodimers has one or more amino acid modifications in the CH1 and/or CL domains, VH and/or VL One or more amino acid modifications within the domain, or combinations thereof. The modified amino acid(s) may typically be part of the interface between the light and heavy chains and are modified to form preferential pairing between the respective heavy chain and the desired light chain, thereby forming the two heavy chains and the two heavy chains of the heterodimer pair. When the canine light chains are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains over the other. Similarly, the heavy chain of the second heterodimer typically pairs with the second light chain preferentially over the first light chain.

Description

Modified antigen-binding polypeptide constructs and uses thereof {MODIFIED ANTIGEN BINDING POLYPEPTIDE CONSTRUCTS AND USES THEREOF}

Cross reference to related applications

This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/003,663, filed May 28, 2014, and U.S. Provisional Patent Application Serial No. 62/154,055, filed April 28, 2015, which is incorporated herein by reference in its entirety for all purposes.

This application is related to PCT/CA2013/050914 (filed on November 28, 2013), US Provisional Application No. 61/730,906 (filed on November 28, 2012), US Provisional Application No. 61/761,641 (filed on February 2013). 6), U.S. Provisional Application No. 61/818,874 (filed May 2, 2013), and U.S. Provisional Application No. 61/869,200 (filed Aug. 23, 2013), the entire disclosures of which are incorporated herein by reference in their entirety. incorporated herein for all purposes).

sequence listing

This application is filed in electronic form in ASCII format and contains a Sequence Listing, incorporated herein by reference in its entirety. Said ASCII copy, created on Friday, May 29, 2015, is referred to as 97993-945204(000110PC)_SL.txt and is 27,012 bytes in size.

A bispecific antibody can bind to at least two different epitopes. The epitopes may be on homologous antigens, or each epitope may be on a different antigen. The properties of bispecific antibodies make them useful tools for a variety of therapeutic applications, where there is a therapeutic benefit of targeting or collecting more than one molecule in the treatment of disease. One approach to forming bispecific antibodies would involve co-expression of two native antibody heavy chains and two native antibody light chains. Since antibody heavy chains have evolved to associate with antibody light chains in a relatively random manner, correctly forming bispecific antibodies in a format similar to wild type remains challenging. As a result of this random pairing, co-expression of two antibody heavy chains and two antibody light chains naturally induces heavy-light chain pairing scrambling. Such mispairing remains a major difficulty for the creation of bispecific therapies, and homologous pairing is an essential requirement for good manufacturability and biological efficacy.

Several approaches have been described for making bispecific antibodies in which a particular antibody light chain or fragment pairs with a particular antibody heavy chain or fragment. A review of various approaches to addressing this problem can be found in Klein et al., (2012) mAbs 4:6, 1-11. International Patent Application No. PCT/EP2011/056388 (WO 2011/131746) discloses that asymmetric mutations are directional "Fab-arm" or "half-arm" between two monospecific IgG4- or IgG4-like antibodies on incubation under reducing conditions. Describes an in vitro method for generating heterodimeric proteins, which are introduced into the CH3 regions of two monospecific initiator proteins to drive a "molecule" exchange.

Schaefer et al. (Roche Diagnostics GmbH) describes a method for assembling two heavy chains and two light chains derived from pre-existing antibodies into a human bivalent bispecific IgG antibody without artificial linkers (PNAS (2011) 108 (27): 11187-11192). The method involves exchanging the heavy and light chain domains in the half 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 them together under specific redox conditions (J. Mol. Biol. ( 2012) 420:204-19).

Zhu et al. (Genentech) engineered a mutant at the VL/VH interface of a diadi construct consisting of a variant domain antibody fragment completely devoid of the constant domain, resulting in a heterodimeric diabody (Protein Science (1997) 6:781- 788). Similarly, Igawa et al. (Chugai) also engineered a mutant at the VL/VH interface of a single-stranded diabody to inhibit conformational isomerization of the diabody and promote selective expression (Protein Engineering, Design & Selection (2010) 23:667- 677).

U.S. Patent Publication No. 2009/0182127 (Novo Nordisk, Inc.) discloses amino acid residues at the Fc interface and CH1:CL interface of light-heavy chain pairs that reduce the ability of one pair of light chains to interact with the other pair of heavy chains. The formation of bispecific antibodies is described by modifying .

US Patent Publication No. 2014/0370020 (Chugai) describes modulating the binding between the CH1 and CL regions of an antibody by substituting amino acids present on the interface between these regions with charged amino acids.

summary

Described herein is an isolated antigen-binding polypeptide construct comprising at least one first heterodimer and a second heterodimer, wherein the first heterodimer comprises a first immunoglobulin heavy chain polypeptide sequence (H1), and a second heterodimer. 1 immunoglobulin light chain polypeptide sequence (L1); 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 distinct from the corresponding H2 or L2 sequences of the second heterodimer, wherein each of H1 and H2 comprises at least one heavy chain variable domain (V _H domain) and a heavy chain constant domain (C _H1 domain); L1 and L2 each comprise at least one light chain variable domain (V _L domain) and a light chain constant domain ( _CL domain); and at least one of H1, H2, L1 and L2 comprises an amino acid modification of at least one of the at least one constant domain and/or at least one variable domain, wherein H1, compared to L2, preferentially pairs with L1 , H2 pairs preferentially with L2, compared to L1.

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

In some embodiments, the at least one amino acid modification is selected from at least one amino acid modification shown in a table or an example.

In some embodiments, H1 pairs preferentially with L1 over L2, and H2 preferentially pairs with L2 over L1 (where H1, H2, L1, and L2 are co-expressed with a cell or mammalian cell). or H1, H2, L1 and L2 are co-expressed in a cell-free expression system, or H1, H2, L1 and L2 are co-produced, or H1, H2, L1 and L2 are co-produced through a redox production method. case).

In some embodiments, at least one of H1, H2, L1 and L2 comprises at least one amino acid modification of a V _H and/or V _L domain and at least one amino acid modification of a C _H1 and/or C _L domain, wherein H1 is Pairs preferentially with L1 over L2, and/or H2 preferentially pairs with L2 over L1.

In some embodiments, when H1 comprises at least one amino acid modification in the C _H1 domain, at least one of L1 and L2 comprises at least one amino acid modification in the C _L domain; When H1 comprises at least one amino acid modification in the V _H domain, then at least one of L1 and L2 comprises at least one amino acid modification in the V _L 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 mutations. In some embodiments, at least one of H1, H2, L1 and L2 is a modification of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of at least one constant domain and/or variable domain. include

In some embodiments, when both L1 and L2 co-express with at least one of H1 and H2, at least one of the H1-L1 and H2-L2 heterodimer pairs, respectively, the corresponding H1-L2 or H2-L1 heterodimer Relative pairing with that of a dimer pair 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, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, greater than 95, 96, 97, 98, or 99%, wherein the relative pairing of the modified H1-L1 or H2-L2 heterodimer pair is equivalent to the corresponding H1-L1 or H2-L2 without at least one amino acid modification. greater than each relative pairing observed in heterodimer pairs.

In some embodiments, the thermal stability, as measured by the melting temperature (Tm) (measured by DSF) of at least one of the first heterodimer and the second heterodimer is, of the corresponding heterodimer without at least one amino acid modification. and within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C of the Tm. In some embodiments, the thermal stability, as measured by at least one melting temperature (Tm) (measured by DSF) of each heterodimer comprising at least one amino acid modification, is determined by that of the corresponding heterodimer without at least one amino acid modification. is within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10° C. of the Tm of In some embodiments, the thermal stability, as measured by the melting temperature (Tm) (measured by DSF) of at least one of each heterodimer comprising at least one amino acid modification, is such that the corresponding heterodimer without at least one amino acid modification within about 0, 1, 2, or 3°C of the Tm of the monomer.

In some embodiments, the affinity of each heterodimer for the antigen to which it binds is greater than the affinity of each unmodified heterodimer for the homologous antigen as measured by surface plasmon resonance (SPR) or FACS. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold.

In some embodiments, at least one of H1 and L1 comprises at least one domain comprising at least one amino acid modification, which results in greater steric complementarity of amino acids when H1 is paired with L1 compared to L2. do. In some embodiments, at least one of H2 and L2 comprises at least one domain comprising at least one amino acid modification, which results in greater steric complementarity of amino acids when H2 is paired with L2 compared to L1. do. In some embodiments, at least one of H1 and L1 comprises at least one domain comprising at least one amino acid modification, which is greater electrostatic complementarity between charged amino acids when H1 is paired with L1 compared to L2. causes In some embodiments, at least one of H2 and L2 comprises at least one domain comprising at least one amino acid modification, which is greater electrostatic complementarity between charged amino acids when H2 is paired with L2 compared to L1. causes

In some embodiments, at least one amino acid modification of is a set of mutations appearing 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 said Fc binds to said first heterodimer and said second heterodimer with or without one or more linkers do.

In some embodiments, 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. In some embodiments, Fc is a heterodimeric Fc. In some aspects, an Fc comprises one or more modifications in at least one of the C _H3 sequences. In some aspects, the dimerized C _H3 sequence is about 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, as determined by DSC. and a melting temperature (Tm) of greater than or equal to 83, 84, or 85°C. In some embodiments, the 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, is a heterodimer formed with greater than 95, 96, 97, 98, or 99% purity; or about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 when expressed or expressed through a single cell. , is a heterodimer formed with greater than 93, 94, 95, 96, 97, 98, or 99% purity. In some aspects, the Fc comprises one or more modifications in at least one of the C _H3 sequences that promote formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc. In some embodiments, the Fc further comprises at least one C _H2 sequence. In some embodiments, the C _H2 sequence(s) of an Fc comprises one or more modifications. In some aspects, an Fc contains one or more modifications to facilitate selective binding of Fc-gamma receptors.

In some embodiments, Fc comprises:

i) a heterodimeric IgG1 Fc having the modification L351Y_F405A_Y407V in said first Fc polypeptide and the modification T366L_K392M_T394W in said second Fc polypeptide;

ii) a heterodimeric IgG1 Fc having the modification L351Y_F405A_Y407V in said first Fc polypeptide and the modification T366L_K392L_T394W in said second Fc polypeptide;

iii) a heterodimeric IgG1 Fc having the modification T350V_L351Y_F405A_Y407V in said first Fc polypeptide and the modification T350V_T366L_K392L_T394W in said second Fc polypeptide;

iv) a heterodimeric IgG1 Fc having the modification T350V_L351Y_F405A_Y407V in said first Fc polypeptide and the modification T350V_T366L_K392M_T394W in said second Fc polypeptide; or

v) a heterodimeric IgG1 Fc having the modification T350V_L351Y_S400E_F405A_Y407V in said first Fc polypeptide and the modification T350V_T366L_N390R_K392M_T394W in said second Fc polypeptide;

In some embodiments, Fc is linked to the heterodimer by one or more linkers, or Fc is linked 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, one or more linkers comprise one or more antibody hinge regions. In some embodiments, one or more linkers include one or more IgG1 hinge regions. In some embodiments, one or more linkers include one or more modifications. In some embodiments, one or more modifications promote selective binding of Fc-gamma receptors.

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, the sequences of H1, H2, L1, and L2 are derived from human sequences.

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

In some embodiments, heterodimers described herein preferentially pair to form a bispecific antibody. For example, in some embodiments, heavy chain polypeptide sequences H1 and H2 comprise full-length heavy chain sequences comprising a heavy chain constant domain (C _H1 domain), a C _H2 domain, and a C _H3 domain. In some embodiments, the percentage of correctly paired heavy and light chains (e.g., H1-L1:H2-L2) within the 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, 81, 82, 83, greater than 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 heavy or light chain or construct described herein. In some embodiments, the polynucleotide or set of polynucleotides is cDNA.

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

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

Pharmaceutical compositions comprising the constructs described herein and a pharmaceutically acceptable carrier are also described. In some embodiments, the composition further comprises 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.

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

Also described herein is a method of 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.

Also described herein is a method of obtaining a construct described herein from host cell culture, the method comprising the steps of: (a) at least one nucleic acid sequence comprising one or more nucleic acid sequences encoding the construct. Obtaining a host cell culture comprising host cells of; and (b) recovering the construct from the host cell culture.

Also described herein is a method of obtaining a construct described herein, comprising the following steps: (a) obtaining H1, L1, H2, and L2; (b) allowing H1 to preferentially pair with L1 over L2 and for H2 to preferentially pair with L2 over L1; and (c) obtaining the construct.

Also described herein includes a method of making a construct described herein: obtaining a polynucleotide or set of polynucleotides encoding at least one construct; 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 an error pair formed on expression of H1, L1, H2, and L2 H1-L2 and determined by evaluating the amount of H1-L1 and H2-L2 heterodimer pairs formed on expression of H1, L1, H2, and L2 compared to H2-L1 heterodimer pairs); selecting a preferred optimal ratio, wherein transfection of at least one host cell with a preferred optimal ratio of the polynucleotide or set of polynucleotides results in expression of the construct; transfecting at least one host cell with an optimal ratio of the polynucleotide or set of polynucleotides; and culturing the at least one host cell to express the construct.

In some embodiments, selection of an optimal ratio is determined by transfection in a transient transfection system. In some embodiments, transfection of at least one host cell with a desired, optimal ratio of a polynucleotide or set 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 is linked to the first heterodimer and the second heterodimer with or without one or more linkers. In some embodiments, Fc is a heterodimer, optionally comprising one or more amino acid modifications.

Also described herein is a computer-readable storage medium storing a dataset comprising: data representing complementary mutations in a first heterodimer (a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain polypeptide sequence (L1)), a second heterodimer (comprising a second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin light chain polypeptide sequence (L2)), wherein H1 and H2 each comprises at least one heavy chain variable domain (V _H domain) and a heavy chain constant domain (C _H1 domain); L1 and L2 each comprise at least one light chain variable domain (V _L domain) and a light chain constant domain ( _CL domain) ); and computer executable code for determining a likelihood that H1 pairs preferentially with L1 over L2, and/or that H2 pairs preferentially with L2 over L1.

Also described herein is a computer implemented method for determining preferential pairing, the method comprising: obtaining a dataset comprising data representative of complementary mutations in a first heterologous a dimer (comprising a first immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain polypeptide sequence (L1)), a second heterodimer (a second immunoglobulin heavy chain polypeptide sequence (H2) and a second immunoglobulin light chain polypeptide sequence) comprising sequence (L2), wherein H1 and H2 each comprise at least one heavy chain variable domain (V _H domain) and a heavy chain constant domain (C _H1 domain); and L1 and L2 each comprise at least one light chain variable domain. (V _L domain) and a light chain constant domain ( _CL domain), and the dataset of complementary mutations includes data representing a subset of the mutations listed in the Tables or Examples); and determining with a computer processor a likelihood that H1 pairs preferentially with L1 over L2 and/or that H2 pairs preferentially with L2 over L1. In some embodiments, the method further comprises generating a construct described herein.

Also described herein is a method of generating a bispecific antigen-binding polypeptide construct, wherein the bispecific construct comprises: a first heterodimer (a first heterodimer from a first monospecific antigen-binding polypeptide); comprising an immunoglobulin heavy chain polypeptide sequence (H1) and a first immunoglobulin light chain polypeptide sequence (L1), a second heterodimer (a second immunoglobulin heavy chain polypeptide sequence (H2) from a second monospecific antigen-binding polypeptide and a second immunoglobulin light chain polypeptide sequence (L2), wherein H1 and H2 each comprise at least one heavy chain variable domain (V _H domain) and a heavy chain constant domain (C _H1 domain); and L1 and L2 are each at least one light chain variable domain (V _L domain) and a light chain constant domain (C _L domain)), the method comprising: one or more complementary mutations from a dataset described herein in the first introducing into the heterodimer and/or a second heterodimer; and co-expressing the first heterodimer and/or the second heterodimer in at least one host cell to produce an expression product comprising the bispecific construct.

In some embodiments, the method further comprises measuring the amount of the bispecific construct in the expression product compared to other polypeptide products in order to select a desirable subset of complementary mutations. In some embodiments, the bispecific construct has greater than 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, greater than 96%, 97%, 98%, or 99%). In some embodiments, the dataset is a dataset described herein. In some embodiments, the method further comprises adding additional amino acid modifications to at least one of H1, H2, L1, or L2 to increase the purity of the bispecific construct compared to other polypeptide products. includes In some embodiments, the construct comprises an Fc comprising at least two C _H3 sequences, wherein the Fc is linked to the first heterodimer and the second heterodimer with or without one or more linkers. In some embodiments, Fc is a heterodimer, optionally comprising one or more amino acid modifications. In some embodiments, an antigen binding polypeptide is an antibody, Fab, or scFv.

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

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

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

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

In some embodiments of the construct, H1 and/or H2 comprises at least one or a set of amino acid modifications at L124, A139 and V190, and L1 and/or L2 comprises amino acids at F116, V133, L135, and S176. at least one or set of variants. In some embodiments, H1 and/or H2 comprises at least one or a set of amino acid modifications selected from L124E, L124R, A139W, A139G, and V190A, and L1 and/or L2 comprises F116A, V133G, L135V, L135W, S176R , and at least one or set of amino acid modifications selected from S176D. In some embodiments, H1 comprises an amino acid modification selected from the group consisting of: L124E and A139W, or a combination thereof; L1 comprises an amino acid modification selected from the group consisting of: F116A, V133G, L135V, and S176R, or a combination thereof; H2 comprises an amino acid modification selected from the group consisting of: L124R, A139G, and V190A, or a combination thereof; and L2 comprises an amino acid modification selected from the group consisting of: V133G, N137K, S174R, L135W, and S176D, or combinations thereof. In some embodiments, H1 comprises the following amino acid modifications: L124E, and A139W; L1 contains the following amino acid modifications: F116A, V133G, L135V, and S176R; H2 contains the following amino acid modifications: L124R A139G, and V190A; and L2 contains the following amino acid modifications: V133G, L135W, and S176D.

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

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

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

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

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

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

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

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

In some embodiments, the construct comprises an amino acid modification selected from: SMCA designs 9561-9095_1, 9561-9095_2, 9121-9373_1, 9121-9373_2, 9116-9349_1, 9116-9349_2, 9134-9521_1 in the tables herein , 9134-9521_2, 9286-9402_1, 9286-9402_2, 9667-9830_1, 9667-9830_2, 9696-9848_1, 9696-9848_2, 9060-9756_1, 9060-9756_2 -9759_2, 9820-9823_1, and 9820-9823_2. In some embodiments, the construct comprises amino acid modifications 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 some embodiments, H1 and/or H2 does not contain an amino acid modification at position Q179. In some embodiments, H1 does not include amino acid modification Q179E and/or H2 does not include amino acid modification Q179K. In some embodiments, L1 does not contain an amino acid modification at position S131. In one embodiment, L1 does not include 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 amino acid modification T180E. In some embodiments, the construct does not comprise a combination of amino acid modifications, wherein H1 comprises Q179E, L1 comprises S131K, H2 comprises Q179K, and L2 comprises T180E.

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

In some embodiments, H1 does not contain an amino acid modification at position Q179. In some embodiments, H1 does not contain an amino acid modification at position Q179K or Q179E. In some embodiments, L1 does not contain amino acid modifications at positions Q160 and/or T180. In one embodiment, L1 does not contain amino acid modifications at positions 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 contain an amino acid modification at position Q179K or Q179E. In some embodiments, L2 does not contain amino acid modifications at positions Q160 and/or Q180. In one embodiment, L2 does not contain amino acid modifications at positions Q160K, Q160E, and/or T180E. In some embodiments, the construct does not comprise a combination of amino acid modifications, wherein H1 comprises Q179K or Q179E, L1 comprises Q160E, Q160K, and/or T180E, and H2 comprises Q179K or Q179E; and L2 includes Q160K, Q160E, and/or T180E.

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

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

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

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

The embodiments described herein are applicable to constructs in both Fab format and whole antibody format.

Figure 1 depicts: D3H44 heavy and light chain amino acid sequences relative to canonical human germline sequences, aligned for variable, constant, and J-region segments (notation in figure: *sequence identity). 1A is a human VH germline subgroup (one representative sequence is shown for each family). Sequence identity based on alignment of D3H44 to VH3 and IGHJ3*02 FIG. 1B is the human kappa VL germline subgroup (one representative sequence is shown from each family). Sequence identity based on alignment of D3H44 to VKI and IGKJ1*01 FIG. 1C is the human lambda VL germline subgroup (one representative sequence is shown from each family). Sequence Identity Based on Alignment of D3H44 to VL1 and IGLJ1*01 Figure 1D depicts human CH1 allele sequences. 1E depicts human kappa and lambda allelic sequences.
Figure 2 depicts a flow chart for computer modeling of designs with preferential heavy-light chain pairing and for identification of key interfacial residues.
3 depicts an exemplary set of H1, L1, H2, L2 chains designed such that H1 pairs preferentially with L1 over L2, and H2 preferentially pairs with L2 over L1. A schematic representation of the 3D crystal structure of the variable region heavy chain and light chain interface is presented. Mutations introduced at this interface achieve electrostatic and steric complementarity to preferentially forming obligate pairs H1-L1 and H2-L2, respectively. On the other hand, there are poor steric and electrostatic mismatches of incorrect pairs that will lead to reduced pairing propensity for mismatched pairs as well as reduced stability.
Figure 4 illustrates a high-level schematic overview of the processing requirements to form bispecific Mab (monoclonal antibodies) and assay conditions required to quantify heavy-light chain pairs. The design goal of a bispecific Mab with high purity (i.e., HL binding with little or no error pairing) is to ensure that the preferential pairing (through the introduction of specific amino acid mutations) of the two native heavy chains to its native mobilizing light chain is rational. It can be achieved by processing with This method is shown schematically, where H1 is engineered to preferentially pair with L1 but not L2. Similarly, H2 has been engineered to preferentially pair with L2 but not L1. Experimental screening of bispecific Mab designs requires assays capable of simultaneous quantification of H1-L1:H1-L2 and H2-L2:H2-L1. These assay requirements can be simplified by assuming that each bispecific Fab arm can be processed independently. In this case, the assay would only need to quantify H1-L1:H1-L2 or H2-L2:H2-L1, but not both simultaneously.
5 provides a schematic showing how heavy and light chains are tagged and how preferential pairing is measured. In this scheme, circles represent cells into which the three constructs are transfected. Expression products are secreted from the cells and the supernatant (SPNT) is flowed over a detection device, in this case an SPR chip. Based on the detection level of the two different tags fusing to the two light chains competing for heavy chain pairing, a quantitative estimate of the preferential pairing of the heavy chain to the two light chains can be estimated.
Figure 6 depicts a box plot showing the average LCCA performance values of paired:mispaired Fab heterodimers with at least 86:14 for each cluster.
Figure 7 shows representative UPLC-SEC profiles for: A) WT Fab heterodimers as well as B) representatively designed Fab heterodimers (H1L1 Fab components of LCCA designs 9735, 9737, and 9740).
Figure 8 depicts the potential heavy chain conjugation products that can be expected when two different light chains are co-expressed as two different heavy chains in a cell. Preferential pairing was measured using SMCA (monoclonal antibody competition assay).
Figure 9 depicts bias/chain utilization preference within a) D3H44/Trastuzumab, b) D3H44/Cetuximab, and c) Trastuzumab/Cetuximab bispecific systems. Chain utilization was measured within different species observed by LC-MS. The x-axis shows the H1:H2:L1:L2 DNA ratio, and the Y-axis shows the corresponding percentage of each strand in different transfection experiments. In the rest of the system, the total H and L chains will represent 25%. A bias towards the use of one light chain is observed across the entire bispecific system.
10 shows representative UPLC-SEC profiles for WT heterodimers as well as engineered heterodimeric antibodies. 10A and 10B refer to D3H44/Trastuzumab WT and 9060-9756_1, respectively. 10C and 10D refer to D3H44/cetuximab WT and 9820-9823_1, respectively. 10E and 10F refer to Trastuzumab/Cetuximab WT and 9696-9848_1, respectively.
Figure 11 shows: % change of correctly paired Fab components in total error paired Fab components with homologous heavy chains for bispecific antibody samples processed per cluster (D3H44/Trastuzumab and D3H44/ H1:L1 across all H1 species for wildtype for cetuximab; change in H2:L2 across all H2 species for wildtype for trastuzumab/trastuzumab) as well as the bispecific antibody of interest to wildtype Percent change in . % change of correctly paired Fab components in total error-paired Fab components using homologous heavy chains versus clusters in a) D3H44/Trastuzumab, c) D3H44/Cetuximab and e) Trastuzumab/Trastuzumab Appears for each system. Percentage change of the bispecific antibody of interest relative to wild type versus cluster is shown for each system within b) D3H44/Trastuzumab, d) D3H44/Cetuximab and f) Trastuzumab/Cetuximab. The % change of correctly paired Fab components in total mispaired Fab components using homologous heavy chains versus clusters across the entire bispecific system is shown in Figure 11, and the percent change of the bispecific antibody of interest relative to wild type versus clusters. is shown in Table 11h. The reported values also include the estimated changes to engineered bispecific antibody samples, where it is noted that the corresponding wild-type construct was not evaluated by SMCA.
12 depicts a method for making bispecific antibodies using a library of obligate mutation pairs provided herein.

Provided herein are antigen-binding polypeptide constructs (also referred to as heterodimer pairs), which may include a first heterodimer and a second heterodimer, each heterodimer comprising an immunoglobulin heavy chain or fragment thereof. and immunoglobulin light chains. Both heterodimers may comprise: 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 1 (CL); one or more amino acid modifications in an immunoglobulin heavy chain variable domain (VH) and one or more amino acid modifications in an immunoglobulin light chain variable domain (VL); or a combination of said amino acid modifications to both the constant and variable domains of said heavy and light chains. The modified amino acids may typically be part of the interface between the light and heavy chains, and are modified to form preferential pairing between the respective heavy chain and the desired light chain, wherein the heavy chain of the first heterodimer differs from one of the light chains to the other. Compare and pair first. Similarly, the heavy chain of the second heterodimer can preferentially pair with the second light chain compared to the first light chain.

As mentioned above, certain combinations of amino acid modifications described herein promote preferential pairing of heavy chains with certain light chains, thereby allowing bispecific monoclonal antibody (Mab) expression to be associated with minimal or limited pairing. occur, and minimize the need to purify the desired heterodimer from undesired or mispaired products. Such heterodimers can exhibit comparable thermal stability to heterodimers that do not contain amino acid modifications, and can also demonstrate binding affinity to antigen that is comparable to heterodimers that do not contain amino acid modifications.

The design of the first and second heterodimers can be used to form bispecific antibodies that target two different therapeutic targets or two separate epitopes (overlapping or non-overlapping) within homologous antigens.

Also provided herein are methods of making heterodimer pairs.

Justice

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 claimed subject matter belongs. In the event that there is a plurality of definitions for terms herein, the definitions in this section shall prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers may change and particular information may come and go on the Internet, but equivalent information may be found by searching the Internet. Reference thereto attests to the availability and public dissemination of such information.

It is to be understood that the foregoing general description and the following detailed description are illustrative and explanatory only and are not intended to limit any claimed subject matter. Unless specifically stated otherwise in this application, the use of the singular includes the plural.

In this description, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, is the value of any integer within the stated range and, if appropriate, a fraction thereof (e.g., tenths and hundredths of an integer). It is understood to include 1) of. As used herein, "about" means ± 10% of the indicated range, value, sequence, or structure, unless otherwise indicated. As used herein, the terms “a” and “an” are to be understood to refer to “one or more” of the listed components, unless indicated otherwise or indicated by context. Use of the alternative (eg, “or”) should be understood to mean either, both, or any combination of these alternatives. As used herein, the terms "comprises" and "comprises" are used synonymously. In addition, individual single chain polypeptides or immunoglobulin constructs derived from the various combinations of structures and substituents described herein are to be understood to be disclosed by this application to the same extent as if each single chain polypeptide or heterodimer were individually recited. It should be. Thus, the selection of specific components to form individual single chain polypeptides or heterodimers is within the scope of the present invention.

Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and treaties, are hereby expressly incorporated by reference in their entirety.

It is to be understood that the methods and compositions described herein are not limited to the specific methods, protocols, cell lines, constructs, and reagents described herein, and thus may vary. Further, the terminology used herein is intended to describe specific embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which scope is understood to be limited only by the appended claims.

All publications and patents mentioned herein are hereby incorporated by reference in their entirety for the purpose of describing and describing the constructs and methodologies described in the publications, which may be used, for example, with the methods, compositions and compounds described herein. is included The publications discussed herein are provided solely for the purpose of their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors described herein have no right to antedate such disclosure by virtue of prior invention or for any other reason.

In this application, amino acid names and atomic names (e.g., N, O, C, etc.) are defined according to the IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atomic names, etc.), Eur. J. Biochem., 138 , 9-37 (1984) and Eur. “Amino acid residue” is intended to label amino acid residues contained in the group consisting primarily of: the 20 naturally occurring amino acids, namely alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D) , glutamic acid (Glu or 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.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. In other words, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. These terms apply 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, these terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

The term "nucleotide sequence" or "nucleic acid sequence" is intended to denote a continuous stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or any combination thereof.

“Cell”, “host cell”, “cell line” and “cell culture” are used interchangeably herein, and it is to be understood that all of these terms include progeny that result from the growth or culture of a cell. “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 amino acid mimetics that function in a manner similar to natural amino acids. The naturally encoded amino acids are 20 common 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 are compounds having the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen-bonded carbon, carboxyl groups, amino groups, and R groups, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulphide. refers to phonium. These analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. References to amino acids include, for example, naturally occurring proteolytic 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, etc.; and chemically synthesized compounds having properties known in the art as being characteristic of amino acids. Examples of non-naturally occurring amino acids include □-methylamino acids (e.g., methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, hydroxy-histidine, homohistidine), additional methylene in the side chain ("homo" amino acids), and amino acids in which a carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (eg, cysteic acid). Incorporation of non-natural amino acids, including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into proteins of the invention can be beneficial in a number of different ways. D-amino acid-containing peptides and the like show increased stability in vitro or in vivo compared to their L-amino acid-containing counterparts. Thus, construction of peptides and the like incorporating D-amino acids can be particularly useful when greater intracellular stability is desired or required. More specifically, D-peptides and the like are resistant to endogenous peptidase and proteases, and thus provide improved bioavailability of the molecule, and extended lifetime in vivo, when these properties are desired. Additionally, D-peptides and the like cannot be efficiently processed for major histocompatibility complex class II-localized presentation to T helper cells, and thus are less likely to induce a humoral immune response in the whole organism.

Amino acids are referred to herein by their commonly known three-letter symbols or one-letter symbols recommended by the Institute [IUPAC-IUB Biochemical Nomenclature Commission]. Nucleotides may likewise be referred to by their commonly accepted one-letter codes.

"Conservatively modified variants" apply to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, where the nucleic acids do not encode amino acid sequences, essentially identical sequences. Due to the degeneracy of the genetic code, multiple 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 any position where an alanine is specified by a codon, that codon can be changed to the corresponding codon described without altering the encoded polypeptide. Such nucleic acid mutations are "silent mutations", which are a type of mutation that are conservative. Every nucleic acid sequence encoding a polypeptide herein also describes every possible silent modification of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to yield functionally identical molecules. Thus, each silent variation of a nucleic acid encoding a polypeptide is latent in each described sequence.

With respect to amino acid sequences, those skilled in the art will understand that individual substitutions, deletions or additions 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 within the encoded sequence, such alteration may be a deletion of an amino acid, an amino acid It will be appreciated that "conservatively modified variants" occur when the addition of , 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. The conservatively modified variants also add to, and do not exclude, the polymorphic variants, interspecies centromeres 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 each contain amino acids that are 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, eg, Creighton, Proteins: Structures and Molecular Properties (W H Freeman &Co.; 2nd edition (December 1993)).

The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are identical. Sequences, when compared and aligned for maximum correspondence over a comparison window or designated region, by quantification using one of the following sequence comparison algorithms (or other algorithms available to those skilled in the art) or by manual alignment and visual inspection, show that they are identical amino acids. Percentages of residues or nucleotides (i.e., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) are “substantially identical”. This definition also refers to the complement of a test sequence. Identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, where not specified, over the entire sequence of a polynucleotide or polypeptide. Polynucleotides encoding polypeptides of the present invention, including centromeres from species other than humans, are screened under stringent hybridization conditions with a labeled probe having a polynucleotide sequence of the present invention or a fragment thereof, and the polynucleotide sequence is It can be obtained by a process comprising the step of isolating full-length cDNA and genomic clones containing. Such hybridization techniques are well known to those skilled in the art.

A derivative, or variant, of a polypeptide is said to share "homology" or "homologous" to the original peptide if the amino acid sequence of the derivative or variant has at least 50% identity to the sequence of 100 amino acids from the original peptide. In certain embodiments, a derivative or variant is at least 75% identical to a peptide or fragment of said peptide having the same number of amino acid residues as the derivative. In certain embodiments, a derivative or variant is at least 85% identical to a peptide or fragment of said peptide having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of a derivative is at least 90% identical to a peptide or fragment of said peptide 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 the peptide or fragment of the peptide having the same number of amino acid residues as the derivative. In certain embodiments, a derivative or variant is at least 99% identical to a peptide or fragment of said peptide 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, separated and/or recovered from a component of the natural cell culture environment. Contaminant components of the natural environment are typically , substances that interfere with diagnostic or therapeutic uses for the heteromultimer, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

In certain embodiments, as used herein, an “isolated” antigen-binding polypeptide construct described herein is a heterodimer or heterodimer that has been identified and separated and/or recovered from a component of its natural cell culture environment. heterodimer pairs comprising pairs or "isolated" heterodimer pairs. Contaminant components of the natural environment are materials that would interfere with diagnostic or therapeutic uses for the heterodimer or antigen-binding polypeptide construct, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

Heterodimeric and antigen-binding polypeptide constructs and heterodimer pairs are generally purified to substantial homogeneity. The phrases "substantially homogeneous", "substantially homogeneous form" and "substantially homogeneous" are used to indicate that a product is substantially free of by-products derived from undesired combinations (eg homodimers). In this context, the species of interest is a heterodimer comprising H1 and L1 (H1-L1), or H2 and L2 (H2-L2). Contaminants are H1 and L2 (H1-L2), or heterodimers containing H2 and L1 (H2-L1), or homodimers containing H1 and L1 or H2 and L2 (if the Fab moiety is paired correctly or not). whether mispaired or not). Substantial homogeneity, expressed in terms of purity, means that the amount of by-products does not exceed 10% of the total LC-MS intensities from all species present in the mixture, e.g., less than 5%, less than 1%, or 0.5%. less than, which means that the percentages reflect the results from mass spectrometry analysis.

The phrase "selectively (or specifically) hybridizes" refers to the ability of a molecule to only a specific nucleotide sequence under stringent hybridization conditions when the sequence is present in a complex mixture (including but not limited to whole cell or library DNA or RNA). refers to the conjugation, double helix, or hybridization of

Each term as understood by one of ordinary skill in the art of antibody technology is given the meaning acquired in the art, unless explicitly defined otherwise herein. Antibodies are known to have variable regions, hinge regions, and constant domains. Immunoglobulin structure and function is reviewed, for example, in Harlow et al, Eds., 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. "Antigen-binding polypeptide construct" is defined as an immunoglobulin gene or immunoglobulin genes Refers to a substantially encoded polypeptide, or one or more fragments thereof, which specifically bind to an analyte (antigen) Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant regions Gene as well as innumerable immunoglobulin variable region genes.Light chain is classified as either kappa or lambda.Heavy chain is classified as gamma, mu, alpha, delta or epsilon, respectively, immunoglobulin isotype, IgG, IgM , IgA, IgD and IgE In turn, antibodies may belong to one of a number of subtypes, for example, an IgG may belong to the IgG1, IgG2, IgG3, or IgG4 subclasses.

An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The term “light chain” includes full-length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain comprises a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is present 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 sufficient variable region sequence to confer binding specificity. A full-length heavy chain comprises a variable region domain, VH and a constant region domain, VL and three constant region domains CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide and the CH domain is at the carboxyl-terminus, where CH3 is closest to the carboxy-terminus of the polypeptide. The heavy chain can be of any isotype, including: IgG (including the IgG1, IgG2, IgG3 and IgG4 subclasses), IgA (including the IgA1 and IgA2 subclasses), IgM and IgE. The term “variable region” or “variable domain” refers to the portion of the light and/or heavy chain of an antibody that is generally responsible for antigen recognition, and typically includes: About the amino-terminus in the heavy chain (VH) 120 to 130 amino acids and about 100 to 110 amino acids terminal amino acids in the light chain (VL). “Complementarity determining regions” or “CDRs” are amino acid sequences that contribute to antigen binding specificity and affinity. “Framework” regions (FRs) can help maintain the proper conformation of the CDRs to facilitate binding between the antigen binding region and the antigen. Structurally, framework regions may be located within an antibody between the CDRs. The various regions typically exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions. CDRs from the two chains of each pair are typically aligned by framework regions, which may allow binding to specific epitopes. From N-terminus to C-terminus, both light and heavy chain variable regions typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The allocation of amino acids into each domain is typically according to the definition of Kabat, a protein sequence of immunological interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) (unless otherwise indicated). In certain embodiments, the antigen-binding polypeptide construct comprises at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, an immunoglobulin domain comprised within an antigen-binding polypeptide construct provided herein is derived from 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 from a heavy chain antibody, eg, a camel antibody. In certain embodiments, an antigen-binding polypeptide construct provided herein is a mammalian antibody, such as a bovine antibody, a human antibody, a camel antibody (single domain and non-single domain), a rodent antibody, a humanized antibody, a non-humanized antibody. at least one immunoglobulin domain from an isolated antibody, a mouse antibody, or any chimeric antibody. In certain embodiments, an antigen-binding polypeptide construct provided herein comprises at least one immunoglobulin domain from an antibody generated from a synthetic library.

A “bispecific” “bispecific” or “bifunctional” antigen binding protein or antibody is a hybrid antigen binding protein that has two different antigen binding sites. Bispecific antigen binding proteins and antibodies are species of multispecific antigen binding protein antibodies. The two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes, which may be on the same or different molecular targets. A “multispecific antigen binding protein” or “multispecific antibody” is one that targets more than one antigen or epitope. A “bivalent antigen-binding protein” or “bivalent antibody” contains two antigen-binding sites. In some instances, the two binding sites have the same antigenic specificities. Bivalent antigen binding proteins and bivalent antibodies may be bispecific (see below). In certain embodiments, a bivalent antibody other than a “multispecific” or “multifunctional” antibody is typically understood to have each of its homologous binding sites.

As used herein, the term “preferential pairing” refers to the pattern of pairing of a first polypeptide and a second polypeptide, e.g., immunoglobulin heavy chains and immunoglobulin light chains, within antigen-binding polypeptide constructs and heterodimer pairs described herein. is used to describe Thus, "preferential pairing" refers to the preferential pairing of a first polypeptide with a second polypeptide when one or more additional, separate polypeptides are present at the same time and the pairing occurs between said first and second polypeptides do. Typically, preferential pairing occurs as a result of modification (eg, amino acid modification) of one or both of the first and second polypeptides. Typically, preferential pairing results in paired first and second polypeptides being the most abundant dimers after pairing has occurred. In the art, an immunoglobulin heavy chain (H1), if co-expressed with different immunoglobulin light chains (L1 and L2), statistically pairs with both light chains, resulting in H1 paired with L1 versus H1 paired with L2. It is known to result in a mixture that is approximately 50:50. In this context, "preferential pairing" means that, for example, if H1 is co-expressed with both L1 and L2, the amount of H1-L1 heavy chain-light chain heterodimers is greater than the amount of H1-L2 heterodimers. will occur between H1 and L1, for example. Thus, in this case, H1 preferentially pairs with L1 compared to L2.

However, it is also known herein that in the context of wild-type bispecific antibodies generated in the two starting antibody systems, there is an inherent bias for the light chain of the antibody system to pair preferentially with the heavy chain of both antibody systems in some cases. do. Thus, when determining the strength of a design in the context of a bispecific antigen-binding construct, it may be necessary to measure the extent of pairing with the design compared to the extent of pairing in a wild-type system. Thus, in one embodiment, a design is considered to exhibit preferential pairing if the amount of bispecific antibody of interest is greater than the amount of bispecific antibody of interest obtained in a wild type system. In another embodiment, a design is considered to exhibit preferential pairing if the amount of pairing in the weaker arm of the antibody is greater than that seen in the wild type system.

Antibody heavy chains pair with antibody light chains and engage or contact each other at one or more “interfaces”. An “interface” includes one or more “contact” amino acid residues in a first polypeptide that interact with one or more “contact” amino acid residues in a second polypeptide. For example, interfaces exist between the CH3 polypeptide sequences 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. An “interface” may be derived from an IgG antibody, such as 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

An antigen-binding polypeptide construct described herein may include a first heterodimer and a second heterodimer, each heterodimer obtained by pairing of an immunoglobulin heavy chain with an immunoglobulin light chain. The structure and organization of the constant and variable domains of immunoglobulin heavy and light chains are well known in the art. An immunoglobulin heavy chain typically comprises one or more variable (VH) domains, three constant domains, CH1, CH2, and CH3. An immunoglobulin light chain typically contains one variable (VL) domain and one constant (CL) domain. Many variations on this typical format can be performed.

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

Each heterodimer of a heterodimer pair is capable of binding specifically to an antigen or epitope. In one embodiment, the immunoglobulin heavy chain and immunoglobulin light chain of each heterodimer comprise one or more modifications from a known antibody, eg a therapeutic antibody. A therapeutic antibody is one effective for treating a disease or disorder in a mammal having or predisposed to have the disease or disorder. Suitable therapeutic antibodies from which each heterodimer can be derived include, but are not limited to: avagovomab, adalimumab, alemtuzumab, aurograb, bafinuzumab, basiliximab, belimumab, beva cizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab, Eve Ritumomab tiuxetan, infliximab, ipilimumab, rumiliximab, mepolizumab, motavizumab, muromonab, micograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab, omalizumab, palivazumab, panitumumab, pertuzumab, ranibaizumab, reslizumab, rituximab, teplizumab, tocilizumab/atlizumab, tositumomab, trastuzumab, ProxiniumTM, RencarexTM, Stekinumab, and Zalutumumab.

In one embodiment, the immunoglobulin heavy chain and/or immunoglobulin light chain of each heterodimer is in a molecule comprising, but not limited to, the proteins listed below, as well as subunits, domains, motifs and epitopes belonging to the proteins listed below. derived or processed from antibodies that bind: renin; growth hormone (including human growth hormone and bovine growth hormone); growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VII, factor VIIIC, factor IX, tissue factor (TF), and von Willebrand factor; anti-clotting factors such as protein C; atrial natriuretic factor; lung surfactant; plasminogen activators such as urokinase or human urine or tissue-type plasminogen activator (t-PA); Bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulation of normal activation of expressed and secreted T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; Müller duct inhibitors; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins such as beta-lactamase; DNase; IgE; cytotoxic T-lymphocyte associated antigen (CTLA) such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); Receptors for: hormones or growth factors such as eg EGFR, VEGFR; interferons such as alpha interferon (alpha-IFN), beta interferon (beta-IFN) and gamma interferon (gamma-IFN); protein A or D; rheumatoid factor; neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6) , or nerve growth factor; platelet-derived growth factor (PDGF); fibroblast growth factors such as AFGF and PFGF; epidermal growth factor (EGF); transforming growth factors (TGFs) 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); des (1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins such as CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD40, CD40L, CD52, CD63, CD64, CD80 and CD147; erythropoietin; osteoinductive factor; immunotoxin; bone morphogenetic protein (BMP); interferons such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs) such as M-CSF, GM-CSF, and G-CSF; interleukins (IL) such as IL-1 to IL-13; TNF-alpha, superoxide dismutase; T-cell receptor; surface membrane proteins; decay accelerating factors; viral antigens such as, for example, AIDS envelope portions, such as gp120; transport proteins; homing receptor; addressin; regulatory proteins; cell junction molecules such as LFA-1, Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM, a4/p7 integrins, and (Xv/p3 integrins, including the following or subunits thereof, integrins Alpha subunits such as CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, alpha7, alpha8, alpha9, alphaD, CD11a, CD11b, CD51, CD11c, CD41, alphaIIb, alphaIELb; Integrin beta subunits such as CD29, CD 18, CD61, CD104, beta5, beta6, beta7 and beta8 Combinations of integrin subunits including but not limited to: alphaVbeta3, alphaVbeta5 and alpha4beta7; apoptosis members of the pathway; IgE; blood group antigen; flk2/flt3 receptor; obesity (OB) receptor; mp1 receptor; CTLA-4; protein C; Eph receptors such as EphA2, EphA4, EphB2, etc; human leukocyte antigen (HLA) such as HLA- DR; complement proteins such as complement receptors CR1, C1Rq and other complement factors such as C3, and C5; glycoprotein receptors such as Gplb.alpha., GPIIb/IIIa and CD200; and any fragments of the above-listed polypeptides.

In an embodiment, the immunoglobulin heavy chain and/or immunoglobulin light chain of each heterodimer comprises one or more modifications from an antibody that specifically binds to a cancer antigen, including but not limited to: ropin receptor), pleiotropin, KS 1/4 pan-carcinoma antigen; ovarian carcinoma antigen (CA125); prostatic acid phosphate; prostate specific antigen (PSA); melanoma-associated antigen p97; melanoma antigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostate specific membrane antigen; Carcinoma Embryonic Antigen (CEA); polymorphic epithelial mucin antigen; human milk fat globules antigen; colorectal tumor-associated antigens such as: CEA, tag-72, CO17-1A, GICA 19-9, CTA-1 and LEA; Burkitt's lymphoma antigen-38.13; CD19; human B-lymphoma antigen-CD20; CD33; melanoma specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor specific transplant type cell-surface antigen (TSTA); virus-derived tumor antigens (including T-antigens of RNA tumor viruses, DNA tumor viruses and envelope antigens); oncofetal antigen-alpha-fetoproteins such as colon CEA, 514 oncofetal trophoblast glycoprotein and bladder tumor oncofetal antigen; differentiation antigens such as human lung carcinoma antigens L6 and L20; fibrosarcoma antigen; human leukemia T cell antigen-Gp37; neoglycoprotein (neoglycoprotein); sphingolipids; breast cancer antigens such as EGFR (epidermal growth factor receptor); NY-BR-16; NY-BR-16 and HER2 antigen (p185HER2); polymorphic epithelial mucin (PEM); malignant human lymphocyte antigen-apo-1; differentiation antigens such as I antigens (found on fetal red blood cells); Primary endoderm I antigen (found on adult erythrocytes) preimplantation embryo; I(Ma) (found in gastric adenocarcinoma); M18, M39 (found in mammary epithelium); SSEA-1 (found in bone marrow cells); VEP8; VEP9; Myl; Va4-D5; D156-22 (found in colorectal cancer); TRA-1-85 (blood type H); SCP-1 (found in testicular and ovarian cancer); C14 (found in colon adenocarcinoma); F3 (found in lung adenocarcinoma); AH6 (found in gastric cancer); Y hapten; Ley (found in embryonic carcinoma cells); TL5 (blood type A); EGF receptor (found on A431 cells); E1 series (blood type B) (found in pancreatic cancer); FC10.2 (found in embryonic carcinoma cells); gastric adenocarcinoma antigen; CO-514 (blood type Lea) (found in adenocarcinoma); NS-10 (found in adenocarcinoma); CO-43 (blood type Leb); G49 (found in the EGF receptor of A431 cells); MH2 (blood type ALeb/Ley) (found in colon adenocarcinoma); 19.9 (found in colon cancer); gastric cancer mucin; T5A7 (found in bone marrow cells); R24 (found in melanoma); 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8 (found in embryonic carcinoma cells) and SSEA-3 and SSEA-4 (4 to 8-cell stage found in the embryo); cutaneous T-cell lymphoma antigen; MART-1 antigen; Sialy Tn (STn) antigen; colon cancer antigen NY-CO-45; lung cancer antigen NY-LU-12 variant A; adenocarcinoma antigen ART1; carcinoma-associated brain-testicular-cancer antigen (neurofetal antigen MA2; antigen of carcinomatous neurons); Neuro-Tumor Abdominal Antigen 2 (NOVA2); hepatocellular carcinoma antigen gene 520; tumor-associated antigen CO-029; Tumor-associated antigens MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4-a, MAGE-4-b and MAGE-4-b X2; Cancer-Testis Antigen (NY-EOS-1) and any fragment of the above-listed polypeptides.

Human antibodies can be grouped 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 the IgA isotype. In another embodiment, the Fc is derived from an IgE isotype. In another embodiment, the Fc is derived from the IgM isotype. In another embodiment, the Fc is derived from the IgD isotype.

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

Each heterodimer of a heterodimer pair is capable of specifically binding an epitope or antigen. In one embodiment, each heterodimer of a heterodimer pair binds a homologous epitope. In another embodiment, the first heterodimer of a heterodimer pair specifically binds an epitope on one antigen and the second heterodimer of a heterodimer pair specifically binds a different epitope on the homologous antigen do. In another embodiment, a first heterodimer of a heterodimer pair specifically binds to an epitope on a first antigen and a second heterodimer of a heterodimer pair binds to an epitope on a second antigen that is different from the first antigen. binds specifically to For example, in one embodiment, a first heterodimer specifically binds tissue factor, while a second heterodimer specifically binds the antigen Her2 (ErbB2), or vice versa. In an alternative embodiment, the first heterodimer specifically binds tissue factor, while the second heterodimer specifically binds EGFR, or vice versa. In still an alternative embodiment, the first heterodimer specifically binds EGFR, while the second heterodimer specifically binds the antigen Her2, or vice versa. In another embodiment, the first heterodimer specifically binds a molecule or cancer antigen described above. In another embodiment, the second heterodimer specifically binds a molecule or cancer antigen described above.

As indicated above, in some embodiments, the immunoglobulin heavy chain and immunoglobulin light chain of each heterodimer undergo one or more modifications, either from known therapeutic antibodies, or from antibodies that bind various target molecules or cancer antigens. include Amino acid and nucleotide sequences of many such molecules are readily available (Note: eg Genbank: AJ308087.1 (humanized anti-human tissue factor antibody D3H44 light chain variable region and CL domain); Genbank: AJ308086 .1 (humanized anti-human tissue factor antibody D3H44 heavy chain variable region and CH1 domain) Genbank: HC359025.1 (Pertuzumab Fab light chain gene module) Genbank: HC359024.1 (Pertuzumab Fab heavy chain gene module); Genbank: GM685465.1 (antibody Trastuzumab (= Herceptin) - wild type; light chain); Genbank: GM685463.1 (antibody Trastuzumab (= Herceptin) - wild type; heavy chain); Genbank: GM685466.1 (antibody Trastuzumab (= Herceptin) - GC-optimized light chain); and Genbank: GM685464.1 (antibody Trastuzumab (= Herceptin) - GC-optimized heavy chain); and Genbank. Each of the Genbank numbers described herein The sequences of are available from the NCBI website as of November 28, 2012, each of which is incorporated herein by reference in its entirety for all purposes.The amino acid and nucleotide sequences for cetuximab are also known herein, See, for example, the Drug Bank website (supported by the Canadian Institutes of Health, Alberta Innovates - Health Solutions, and the Metabolomics Innovation Center (TMIC), accession number DB00002).

In some embodiments, the isolated antigen-binding polypeptide construct has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 amino acid sequences or fragments thereof set forth in the Tables or Accession Numbers reviewed herein. , 99, or 100% homologous amino acid sequences. In some embodiments, the isolated antigen-binding polypeptide construct has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 amino acid sequences or fragments thereof set forth in the Tables or Accession Numbers reviewed herein. , 99, or 100% homologous polypeptide.

Amino Acid Modifications to Immunoglobulin Heavy and Light Chains

At least one of the heterodimers of the heterodimer pair may contain one or more amino acid modifications to their immunoglobulin heavy chain and/or immunoglobulin light chain, such that the first heterodimer is composed of one light chain and the other light chain. Pairing takes precedence. Similarly, the heavy chain of the second heterodimer can preferentially pair with the second light chain compared to the first light chain. Preferential pairing of one heavy chain with one of two light chains can be based on a design set comprising: one immunoglobulin heavy chain and two immunoglobulin light chains (referred to as the LCCA design set), herein When a globulin heavy chain co-expresses with both immunoglobulin light chains, the immunoglobulin light chain pairs preferentially with one of the two immunoglobulin light chains over the other). Thus, an LCCA design set may 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 pairings exemplified in the LCCA design set are constructed by modifying one or more amino acids that are part of the interface between light and heavy chains. In one embodiment, the exemplified preferential pairing within the LCCA design set is constructed by modifying at least one of 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. one or more amino acids.

In one embodiment, the one or more amino acid modifications are, but are not limited to: conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains (when labeled by Kabat numbering of residues). For example, Almagro [Frontiers In Bioscience (2008) 13: 1619-1630] provides a definition of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

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 interface can be achieved based on steric and hydrophobic contacts, electrostatic/charged interactions, and combinations of various interactions. Complementarity between protein surfaces has been extensively described in the literature in terms of: lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which It implies the nature of the structural and chemical matching between the two interacting surfaces. In one embodiment, at least one of the heterodimers comprises one or more mutations, wherein the immunoglobulin light chain and the mutations introduced into the immunoglobulin light chain introduce new hydrogen bonds across the light and heavy chains at the interface. In one embodiment, at least one of the heterodimers comprises one or more mutations, wherein the immunoglobulin light chain and the mutations introduced into the immunoglobulin light chain introduce new salt bridges across the light and heavy chains at the interface.

Non-limiting examples of a set of suitable LCCA designs 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 main, and a first immunoglobulin light chain without any amino acid modification in the CL domain. 2 contains immunoglobulin light chains. In another embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 1 amino acid modification in the CH1 domain, a first immunoglobulin light chain having at least 1 amino acid modification in the CL domain, and at least 1 amino acid modification in the CL domain. A second immunoglobulin light chain with In another embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 1 amino acid modification in the CH1 domain, a first immunoglobulin light chain having at least 2 amino acid modifications in the CL domain, and at least 2 amino acid modifications in the CL domain. A second immunoglobulin light chain with In another embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 1 amino acid modification in the CH1 domain, a first immunoglobulin light chain having at least 2 amino acid modifications in the CL domain, and at least 1 amino acid modification in the CL domain. A second immunoglobulin light chain with

In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain without an amino acid modification in the CH1 domain, a first immunoglobulin light chain without an amino acid modification in the CL domain, and a second immunoglobulin light chain with at least one amino acid modification in the CL domain. include In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain without amino acid modifications in the CH1 domain, a first immunoglobulin light chain without amino acid modifications in the CL domain, and a second immunoglobulin light chain with at least two amino acid modifications in the CL domain. include

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 without amino acid modifications in the CL domain, and a second immunoglobulin light chain having at least one amino acid modification in the CL domain. Contains globulin light chains. 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 1 amino acid modification in the CL domain, and at least 1 amino acid modification in the CL domain. A second immunoglobulin light chain. 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 1 amino acid modification in the CL domain, and at least 2 amino acid modifications in the CL domain. A second immunoglobulin light chain. 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 2 amino acid modifications in the CL domain. A second immunoglobulin light chain. 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 2 amino acid modifications in the CL domain. A second immunoglobulin light chain.

In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least three amino acid modifications in the CH1 domain, a first immunoglobulin light chain without amino acid modifications in the CL domain, and a second immunoglobulin light chain having at least one amino acid modification in the CL domain. Contains globulin light chains. 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 1 amino acid modification in the CL domain, and at least 1 amino acid modification in the CL domain. A second immunoglobulin light chain. 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 2 amino acid modifications in the CL domain. A second immunoglobulin light chain. 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 3 amino acid modifications in the CL domain. A second immunoglobulin light chain. In one embodiment, the exemplified preferential pairing within the LCCA design set is constructed by modifying at least one of the VH domain of an immunoglobulin heavy chain, the VL domain of a first immunoglobulin light chain, and the VL domain of a second immunoglobulin light chain. one or more amino acids. Non-limiting examples of a set of suitable LCCA designs are shown in the tables 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 a second immunoglobulin light chain with at least one amino acid modification in the VL domain. include In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain without amino acid modifications in the VH domain, a first immunoglobulin light chain without amino acid modifications in the VL domain, and a second immunoglobulin light chain with at least two amino acid modifications in the VL domain. include

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 a second immunoglobulin chain having at least one amino acid modification in the VL domain. Contains globulin light chains. In one embodiment, the LCCA design set comprises an immunoglobulin heavy chain having at least 1 amino acid modification in the VH domain, a first immunoglobulin light chain having at least 1 amino acid modification in the VL domain, and at least 1 amino acid modification in the VL domain. A second immunoglobulin light chain. 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 two amino acid modifications in the VL domain. A second immunoglobulin light chain.

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 modifications in the VL domain, and a second immunoglobulin chain having at least one amino acid modification in the VL domain. Contains globulin light chains. 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 two amino acid modifications in the VL domain, and at least one amino acid modification in the VL domain. A second immunoglobulin light chain. 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 one amino acid modification in the VL domain. A second immunoglobulin light chain.

In one embodiment, the LCCA design set can be combined to provide a combination comprising two separate immunoglobulin heavy chains (H1 and H2) and two separate immunoglobulin light chains (L1 and L2), wherein H1, H2, L1 , and L2 are co-expressed, H1 preferentially pairs with L1, and H2 preferentially pairs with L2. In some embodiments, amino acid modifications described herein are in the context of bispecific antibody constructs. For example, the set of designs 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 within the Fc region, as described in the Examples.

Mobility of certain amino acid modifications identified herein relative to other antibodies

Although specific amino acid modifications to the above-identified immunoglobulin heavy and light chains have been described with respect to the D3H44 anti-tissue factor extracellular domain antibody, trastuzumab, and cetuximab immunoglobulin heavy and light chains, these amino acid modifications have a similar pattern to other It is contemplated and exemplified that being able to migrate into immunoglobulin heavy and light chains, resulting in preferential pairing of one immunoglobulin heavy chain with one of two immunoglobulin light chains (see Examples, Figures, and graph).

The VH:VL and CH1:CL interface residues within the interface between immunoglobulin heavy and light chains are relatively well conserved (Padlan et al., 1986, Mol. Immunol. 23(9): 951-960). This sequence conservation increases the likelihood that functionally active antibody binding domains will form during combinatorial pairing of light and heavy chains, as a result of innovative constraints. As a result of this sequence conservation, the sequence modifications driving preferential pairing in the specific example noted above for D3H44 approximate the results obtained for preferential pairing, as this region exhibits high sequence conservation in antibodies. according to the fact that it can be transferred to other heavy and light chain pair heterodimers with equivalent results; Additionally, when sequence differences do not occur, they are generally distal to the CH1:CL interface. This is especially the case for the CH1 and CL domains. However, there is some sequence variability in the antigen-binding site with respect to CDR (complementarity-determining region) loop residues (and lengths), particularly for CDR-H3. Thus, in one embodiment, a heterodimer pair described herein comprises a heterodimer, wherein when the amino acid sequence of the antigen-binding site differs significantly from that of the D3H44 antibody, at least one heterodimer comprises a CDR loop one or more amino acid modifications within the distal VH and/or VL domains. In another embodiment, a heterodimer pair described herein comprises a heterodimer, wherein when the amino acid sequence of the antigen-binding site is substantially homologous to that of the D3H44 antibody, at least one heterodimer comprises a CDR loop one or more amino acid modifications within the VH and/or VL domains, either proximal or distal.

In one embodiment, the amino acid modifications described herein are transferable to immunoglobulin heavy and light chains of antibodies based on human or humanized IgG1/κ. 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 using the commonly used VH and VL subgroups. Non-pharmaceutical examples of such antibodies include Pertuzumab.

In one embodiment, the amino acid modifications described herein are transferable to immunoglobulin heavy and light chains of antibodies with frameworks in germline proximity. An example of such an antibody includes obinutuzumab.

In one embodiment, the amino acid modifications described herein are transferable to immunoglobulin heavy and light chains of an antibody having VH:VL interdomain angles close to the average observed for heavy and light chain pairs. Examples of antibodies include, but are not limited to, Pertuzumab. In another embodiment, the amino acid modifications described herein are transferable to immunoglobulin heavy and light chains of antibodies having canonical CL and CH1 domains. A suitable example of such an antibody includes, but is not limited to, Trastuzumab.

In some embodiments, specific subsets of amino acid modifications described herein are utilized within variant domains within the antigen-binding constructs provided above.

The Examples, Figures, and Tables illustrate the following: Amino acid modifications (e.g., within one or more Fab fragments comprising variable and constant regions) can be transferred to other immunoglobulin heavy and light chains, resulting in a single immunoglobulin. It causes a similar pattern of preferential pairing with one of the heavy chain's two immunoglobulin light chains.

preferential pairing

As described above, at least one heterodimer of an antigen-binding construct/heterodimer pair described herein may comprise one or more amino acid modifications to its immunoglobulin heavy chain and/or immunoglobulin light chain, whereby The heavy chain of one heterodimer, e.g. H1, preferentially pairs with one of the light chains, e.g. L1, over the other one of the light chains, e.g. L2, and the heavy chain of the other heterodimer, For example, H2 preferentially pairs with one of the light chains, eg L2, over the other of the light chains, eg L1. In other words, desired, preferential pairing is considered to exist between H1 and L1 and between H2 and L2. For example, preferential pairing between H1 and L1 is, if H1 is combined with L1 and L2, compared to respective pairings of corresponding H1/L1 to H2/L2 pairs without one or more amino acid modifications. , is considered to occur if the yield of H1-L1 heterodimers is greater than the yield of mispaired H1-L2 heterodimers. Similarly, preferential pairing between H2 and L2 occurs when H2 is combined with L1 and L2, compared to pairing with each of the corresponding H1-L1 to H2-L2 pairs without at least one amino acid modification. , is considered to occur if the yield of H2-L2 heterodimers 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, correctly paired obligate pairs, or heterodimers of interest herein. , whereas heterodimers comprising H1 and L2 (H1-L2), or H2 and L1 (H2-L1) are referred to herein as mispaired heterodimers. The set of mutations corresponding to two heavy chains and two light chains that are designed to achieve selective pairing of H1-L1 and H2-L2 is referred to as a design set.

Thus, in one embodiment, when one immunoglobulin heavy chain of a heterodimer is co-expressed 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 co-expressed 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 co-expressed 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 co-expressed 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 co-expressed with two immunoglobulin light chains, the relative yield of the desired heterodimer is greater than 90%. In another embodiment, when one heavy immunoglobulin chain of a heterodimer is co-expressed with two light immunoglobulin chains, the relative yield of the desired heterodimer is greater than 95%.

In the above example, preferential pairing between H1-L1 occurs when H1 co-expresses with L1 and L2, when the amount of the desired H1-L1 heterodimer is greater than the amount of mispaired H1-L2 heterodimer. It is considered to do Similarly, preferential pairing between H2-L2 occurs when H2 co-expresses with L1 and L2, when the amount of the desired H2-L2 heterodimer is greater than the amount of mispaired H2-L2 heterodimer. is considered to be Thus, in one embodiment, when one immunoglobulin heavy chain of a heterodimer is co-expressed with two immunoglobulin light chains, the ratio of the relative yield of the desired heterodimer to mispaired heterodimer is greater than 1.25:1. am. In one embodiment, when one immunoglobulin heavy chain of a heterodimer is co-expressed with two immunoglobulin light chains, the ratio of the relative yield of the desired heterodimer to mispaired heterodimer is greater than 1.5:1. In another embodiment, when one immunoglobulin heavy chain of the heterodimer is co-expressed with two immunoglobulin light chains, the ratio of the relative yield of the desired heterodimer to mispaired heterodimer is greater than 2:1. . In another embodiment, when one immunoglobulin heavy chain of the heterodimer is co-expressed with three immunoglobulin light chains, the ratio in relative yield of the desired heterodimer to mispaired heterodimer is greater than 3:1. . In another embodiment, when one heavy immunoglobulin chain of the heterodimer is co-expressed with five light immunoglobulin chains, the ratio of the relative yield of the desired heterodimer to mispaired heterodimer is greater than 5:1. . In another embodiment, when one immunoglobulin heavy chain of the heterodimer is co-expressed with 10 immunoglobulin light chains, the ratio of the relative yield of the desired heterodimer to mispaired heterodimer is greater than 10:1. . In another embodiment, when one immunoglobulin heavy chain of the heterodimer is co-expressed with 25 immunoglobulin light chains, the ratio of the relative yield of the desired heterodimer to mispaired heterodimer is greater than 25:1. . In another embodiment, when one immunoglobulin heavy chain of the heterodimer is co-expressed with 50 immunoglobulin light chains, the ratio of the relative yield of the desired heterodimer to mispaired heterodimer is greater than 50:1. .

In some embodiments, heterodimers described herein preferentially pair to form a bispecific antibody. In some embodiments, the construct comprises heterodimers that preferentially pair to form a bispecific antibody selected from D3H44/Trastuzumab, D3H44/Cetuximab, and Trastuzumab/Cetuximab. In some embodiments, the bispecific antibody comprises 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 yield the following 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 correctly paired bispecific antibody species. In some embodiments, DNA ratios are selected to yield the maximum amount of correctly 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 compared to all species is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (see, e.g., Tables 29a-29c and 30a-30c ). In some embodiments, the percentage of correctly paired bispecific species obtained by co-expression of the corresponding wild-type H1, H2, L1 and L2 without amino acid modifications described in Tables 28a-28c greater than the percentage of species. In some embodiments, the percentage of correctly paired bispecific species is the number of correctly paired bispecific species obtained by co-expression of wildtype H1, H2, L1 and L2 without amino acid modifications described in Tables 28a-28c. percentage by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 75%.

Thermal stability of heterodimers

In addition to promoting preferential pairing, amino acid substitutions were selected so that the mutations did not destabilize the Fab heterodimer. Thus, in most cases, stability measurements of Fab heterodimers were very close to those of wild-type Fab (eg, within 3° C. of wild-type Fab).

Thus, in some embodiments, each heterodimer of a pair of heterodimers described herein comprises homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It has the same or comparable thermal stability to that of the heterodimer. In one embodiment, thermal stability is measured by measuring the melting temperature, or Tm. Thus, in one embodiment, the thermal stability of a heterodimer described herein is determined by a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It is within about 10 ℃ of that of. Thus, in one embodiment, the thermal stability of a heterodimer described herein is determined by a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. is within about 5°C of that of In another embodiment, the thermal stability of the heterodimers described herein is determined by a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. within about 3°C of that. In another embodiment, the thermal stability of the heterodimers described herein is determined by a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. within about 2°C of that. In another embodiment, the thermal stability of the heterodimers described herein is determined by a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It is within about 1.5 ℃ of that. In another embodiment, the thermal stability of the heterodimers described herein is determined by a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It is within about 1 degree Celsius of that. In another embodiment, the thermal stability of the heterodimers described herein is determined by a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It is within about 0.5 ℃ of that. In another embodiment, the thermal stability of the heterodimers described herein is determined by a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It is within about 0.25 ℃ of that.

Moreover, in some embodiments, the thermal stability of heterodimers described herein is such that heterodimers comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. significantly improve (i.e., increase) compared to that of Thus, in one embodiment, the thermal stability of a heterodimer described herein is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8. 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 (heterodimers comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein compared to).

Affinity of the heterodimer for the antigen

In one embodiment, each heterodimer of the heterodimer pair is relative to that of the heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. have homologous or comparable affinities for their respective antigens. In one embodiment, the heterodimer of the heterodimer pair is about 50% of that of a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. The embryo has an affinity for its respective antigen. In one embodiment, the heterodimer of the heterodimer pair is about 25% of that of the heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. The embryo has an affinity for its respective antigen. In one embodiment, the heterodimer of the heterodimer pair is about 10 of that of a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. The embryo has an affinity for its respective antigen. In another embodiment, the heterodimer of the heterodimer pair is about one of the heterodimers comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It has an affinity for its respective antigen that is 5-fold. In another embodiment, the heterodimer of the heterodimer pair is about one of the heterodimers comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It has an affinity for its respective antigen that is 2.5 fold. In another embodiment, the heterodimer of the heterodimer pair is about one of the heterodimers comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It has an affinity for its respective antigen that is 2-fold. In another embodiment, the heterodimer of the heterodimer pair is about one of the heterodimers comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It has an affinity for its respective antigen that is 1.5 fold. In another embodiment, the heterodimer of the heterodimer pair is substantially identical to that of a heterodimer comprising homologous immunoglobulin heavy and light chains without amino acid modifications to the CH1, CL, VH, or VL domains described herein. It has an affinity for each antigen of its homology.

Additional Optimal Variations

In one embodiment, the immunoglobulin heavy and light chains of the heterodimeric pairs described herein are further modified (i.e., by covalent attachment of various types of molecules) such that the covalent attachment forms preferential pairing between the heavy and light chains. interfere with, affect the ability of the heterodimer to bind to its antigen, or affect its stability. For example, but not limited to, such modifications include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, cell ligands or other proteins. Include connections. Any of a myriad of chemical transformations 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 immunoglobulin heavy and light chains of a heterodimeric pair described herein can be conjugated (directly or indirectly) to a therapeutic agent or drug moiety that modifies a given biological response. A therapeutic agent or drug moiety should be construed as not limited to traditional chemotherapeutic agents. For example, a drug moiety can be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, onconase (or another cytotoxic RNase), Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as 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 ( Reference, International Publication No. WO 97/33899), AIM II (reference, International Publication No. WO 97/34911), Fas ligand (Takahashi et al., 1994, J. Immunol., 6:1567), and VEGI ( See International Publication No. WO 99/23105), thrombotic or anti-angiogenic agents such as angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines (e.g., 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 (eg, growth hormone ("GH")).

Moreover, in an alternative embodiment, the antibody may be conjugated to a therapeutic moiety useful for conjugation to a radiometal ion, such as a radioactive substance or a macrocyclic chelator (see above for examples of radioactive substances). In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA), which is via a linker molecule the antibody can be attached to Such linker molecules are commonly 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 immunoglobulin heavy and light chains of the heterodimer are displayed as fusion proteins comprising tags to facilitate purification and/or testing, and the like. As referred to herein, a "tag" is any added set of amino acids provided within a protein that contributes to the identification and purification of the protein, whether C-terminal, N-terminal, or internal. Suitable tags include, but are not limited to: tags known to those skilled in the art that will be useful for purification and/or testing, such as albumin binding domain (ABD), His tag, FLAG tag, glutathione-s-transferase , hemagglutinin (HA) and maltose binding domains. The tagged protein may also be engineered to include a cleavage site, such as a thrombin, enterokinase or factor X cleavage site, to facilitate removal of the tag before, after, or during purification.

In some embodiments, one or more of the cysteine residues at the bottom of the Fab domain in the light chain (position 214, Kabat numbering) and heavy chain (position 233, Kabat numbering) forming interchain disulfide bonds are serine or alanine or non-cysteine or individual Can be transformed into amino acids.

It is contemplated that additional amino acid modifications may be made to the immunoglobulin heavy chain to increase the level of preferential pairing and/or thermal stability of the heterodimeric pair. For example, additional amino acid modifications can be made to the immunoglobulin heavy chain Fc domain to drive preferential pairing between heterodimeric pairs compared to homodimeric pairs. Such amino acid modifications are known in the art and include, for example, those described in US Patent Publication No. 2012/0149876. Alternatively, alternative strategies for driving preferential pairing between heterodimer pairs compared to homodimer pairs, such as “knobs into holes”, charged residues with ionic interactions, and Strand-exchange processing domain (SEED) technology can also be used. This last strategy is described in the art and is reviewed in Klein et al , supra. A further description of the Fc domain follows.

Fc domain

In an embodiment, the antigen-binding polypeptide construct comprises a full-length immunoglobulin heavy chain, and the construct will comprise an Fc. In some embodiments, an Fc comprises at least one or two CH3 domain sequences. In some embodiments, the antigen-binding polypeptide construct comprises a heterodimer comprising only a Fab region of a heavy chain, wherein the Fc, with or without one or more linkers, comprises a first heterodimer and/or a second heterodimer join in In some embodiments the Fc is a human Fc. In some embodiments, the Fc is a human IgG or IgG1 Fc. In some embodiments, Fc is a heterodimeric Fc. In some embodiments, an Fc comprises at least one or two CH2 domain sequences.

In some aspects, an Fc comprises one or more modifications in at least one of the C _H3 domain sequences. In some aspects, an Fc comprises one or more modifications in at least one of the C _H2 domain sequences. In some embodiments, Fc is a single polypeptide. In some embodiments, Fc is multiple peptides, such as two polypeptides.

In some aspects, an Fc comprises one or more modifications in at least one of the C _H3 sequences. In some aspects, an Fc comprises one or more modifications in at least one of the C _H2 sequences. In some embodiments, Fc is a single polypeptide. In some embodiments, Fc is multiple peptides, such as two polypeptides.

In some embodiments, the Fc is an Fc described below: patent application PCT/CA2011/001238 filed on November 4, 2011 or PCT/CA2012/050780 filed on November 2, 2012 (the full disclosure of which is incorporated herein by reference in its entirety).

In some aspects, the constructs described herein include a heterodimeric Fc comprising an asymmetrically modified modified CH3 domain. A heterodimeric Fc will comprise two heavy chain constant domain polypeptides, which can be used interchangeably: a first heavy chain polypeptide and a second heavy chain polypeptide, as long as the Fc comprises one first heavy chain polypeptide and one second heavy chain polypeptide. can Generally, a first heavy chain polypeptide comprises a first CH3 sequence and a second heavy chain polypeptide comprises a second CH3 sequence.

Two CH3 sequences containing one or more amino acid modifications introduced in an asymmetric fashion will generally result in a heterodimeric Fc rather than a homodimer when the two CH3 sequences dimerize. As used herein, "asymmetric amino acid modification" refers to any modification in which an amino acid at a specific position on a first CH3 sequence differs from an amino acid on a second CH3 sequence at the same position, wherein the first and second CH3 sequences preferentially pair It forms heterodimers rather than homodimers. Said heterodimerization is characterized by modification of only one of the two amino acids at the same respective amino acid position on each sequence; Or it may be the result of modification of both amino acids on each sequence at the same position in each of the first and second CH3 sequences. The first and second CH3 sequences of the heterodimer Fc may contain one or more asymmetric amino acid modifications.

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

Typically an Fc may contain two adjacent heavy chain sequences (A and B) capable of dimerization. In some embodiments, the sequence of one or both of the Fc comprises one or more mutations or modifications in: L351, F405, Y407, T366, K392, T394, T350, S400, and/or N390 (using EU numbering) . In some embodiments, an Fc comprises a mutant sequence shown in Table X. In some embodiments, Fc comprises mutants of variant 1 A-B. In some embodiments, Fc comprises mutants of variant 2 A-B. In some embodiments, Fc comprises mutants of variant 3 A-B. In some embodiments, Fc comprises mutants of variant 4 A-B. In some embodiments, Fc comprises mutants of variant 5 A-B.

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

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

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

In one embodiment, the heterodimeric Fc is a modified CH3 domain comprising a first CH3 sequence with amino acid modifications at positions L351, F405 and Y407, and a second CH3 sequence with amino acid modifications at positions T366, K392, and T394. wherein one or both of the CH3 sequences further comprises the amino acid modification T350V.

In one embodiment, the heterodimeric Fc comprises a modified CH3 domain comprising the following amino acid modifications, wherein "A" represents an amino acid modification to a first CH3 sequence and "B" to a second CH3 sequence. 대한 아미노산 변형을 나타낸다: 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, 및/또는 B :T350V_T366L_N390R_K392M_T394W.

One or more asymmetric amino acid modifications can promote the formation of a heterodimeric Fc, wherein the heterodimeric CH3 domain has comparable stability to a wild-type homodimeric CH3 domain. In an embodiment, the one or more asymmetric amino acid modifications promote formation of a heterodimeric Fc domain, wherein the heterodimeric Fc domain has comparable stability to a wild-type homodimeric Fc domain. In an embodiment, the one or more asymmetric amino acid modifications promote formation of a heterodimeric Fc domain, wherein the heterodimeric Fc domain has a stability observed via melting temperature (Tm) in a differential scanning calorimetry study, wherein the melting temperature is It is within 4°C of that observed for the corresponding symmetric wild-type homodimeric Fc domain. In some aspects, the Fc comprises one or more modifications in at least one of the C _H3 sequences that promote formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc.

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

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

Additional methods for modifying monomeric Fc polypeptides to promote heterodimer Fc formation are described in International Application Publication No. WO 96/027011 (knobs into holes), Gunasekaran et al. (Gunasekaran K. et al. (2010) J Biol Chem. 285, 19637-46, electrostatic design to achieve selective heterodimerization), Davis et al. (Davis, JH. et al. (2010) Prot Eng Des Sel ;23(4): 195-202, strand exchange processing domain (SEED) technology), and Labrijn et al [Efficient generation of stable 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, Verploegen S, Kundu A, Gramer MJ, van Berkel PH, van de Winkel JG, Schuurman J, Parren PW. Proc Natl Acad Sci USA. 2013 Mar 26;110(13):5145-50.

In some embodiments, an isolated construct described herein is an antigen-binding construct that binds an antigen; and dimeric Fc polypeptide constructs that have superior biochemical properties, such as stability and ease of manufacture, compared to antigen-binding constructs that do not contain the homologous Fc polypeptide. A number of mutations within the heavy chain sequence of Fc to selectively alter the affinity of antibody Fc for different Fcgamma receptors are known in the art. In some aspects, an Fc contains one or more modifications to facilitate selective binding of Fc-gamma receptors.

The CH2 domain is amino acids 231-340 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); F243L/R292P/Y300L/V305I/P396L, F243L/R292P/Y300L/L235V/P396L (Stavenhagen JB, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep 15;67(18):8882-90; Nordstrom JL , Gorlatov S, Zhang W, et al. Breast Cancer Res. 2011 Nov 30;13(6):R123); F243L (Stewart R, Thom G, Levens M, et al. Protein Eng Des Sel. 2011 Sep;24(9):671-8.), S298A/E333A/K334A (Shields RL, Namenuk AK, Hong K, et al J Biol Chem. 2001 Mar 2;276(9):6591-604); S239D/I332E/A330L, S239D/I332E (Lazar GA, Dang W, Karki S, et al. Proc Natl Acad Sci US A. 2006 Mar 14;103(11):4005-10); S239D/S267E, S267E/L328F (Chu SY, Vostiar I, Karki S, et al. Mol Immunol. 2008 Sep; 45(15):3926-33); S239D/D265S/S298A/I332E, S239E/S298A/K326A/A327H, G237F/S298A/A330L/ I332E, S239D/I332E/S298A, S239D/K326E/A330L/I332E/S298A, G236A/S239D/D270L/I332E, S239E/ S267E/H268D, L234F/S267E/N325L, G237F/V266L/S267D and other mutations listed in WO2011/120134 and WO2011/120135, incorporated herein by reference. Mutations are listed on page 283 of Therapeutic Antibody Engineering (William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012).

In some embodiments, a CH2 domain comprises one or more asymmetric amino acid modifications. In some embodiments, a CH2 domain comprises one or more asymmetric amino acid modifications to promote selective binding of an FcγR. In some embodiments, the CH2 domain allows for isolation and purification of an isolated construct described herein.

FcRn binding and PK parameters

As is known in the art, binding to FcRn recirculates the translocated antibody from endosomes back into the blood stream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al. ., 2000, Annu Rev Immunol 18:739-766). This procedure results in favorable antibody serum half-lives ranging from 1 to 3 weeks, with exclusion of renal filtration due to the large size of the full-length molecule. Binding of Fc to FcRn also plays an important role in antibody transport. Thus, in one embodiment, constructs of the invention are capable of binding FcRn.

Additional Modifications to Improve Effector Function

In some embodiments, a construct described herein may be modified to improve its effector function. Such modifications are known in the art and include: afucosylation or engineering of the affinity of the Fc portion of the antibody for activating receptors for ADCC, mainly FCGR3a, or C1q for CDC. Table Y below summarizes the various designs reported in the literature on effector function processing.

Thus, in one embodiment, the constructs described herein may include a dimeric Fc comprising one or more amino acid modifications noted in the table above that confer improved effector function. In another embodiment, the construct can be afucosylated to improve effector function.

linker

A construct described herein may include one or more heterodimers described herein operably linked to an Fc described herein. In some embodiments, Fc is linked to one or more heterodimers with or without one or more linkers. In some embodiments, Fc is directly linked to one or more heterodimers. In some embodiments, Fc is linked to one or more heterodimers by one or more linkers. In some embodiments, 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, one or more linkers comprise one or more antibody hinge regions. In some embodiments, one or more linkers include one or more IgG1 hinge regions.

Methods of making heterodimer pairs

As referred to herein, a heterodimer pair may include a first heterodimer and a second heterodimer, each heterodimer having at least one VH and CH1 domain, or a fragment thereof, of an immunoglobulin heavy chain. 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. Standard techniques such as [Sambrook and Russell, Molecular Cloning:

A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2nd ed., 1989); Short Protocols in Molecular Biology (Ausubel et al., John Wiley and Sons, New York, 4th ed., 1999); and Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, D.C., 2nd ed., 1998) are recombinant nucleic acid methods, nucleic acid synthesis, cell culture, transgene introduction, and recombinant proteins. can be used for expression. The heterodimers and heterodimer pairs described herein can be chemically synthesized.

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

For example, methods for expressing and co-expressing immunoglobulin heavy and light chains in a host cell are well known in the art. Additionally, methods for tagging heavy and/or light chains using recombinant DNA techniques are also well known in the art. Expression vectors and host cells suitable for expression of heavy and light chains are also well known in the art, as described below.

Methods for generating bispecific antibodies that do not rely solely on use in monoclonal or transient cell lines expressing all four chains are known in the art (Gramer, et al. (2013) mAbs 5, 962; Strop et al. (2012) J Mol Biol 420, 204.). This method relies on post-production arm exchange under redox conditions of the two pairs of light and heavy chains involved in the formation of the bispecific antibody (redox production). In this approach, H1:L1 and H2:L2 pairs can be expressed in two different cell lines to independently generate two Fab arms. Subsequently, the two Fab arms are mixed under selected redox conditions to achieve recombination of the two native heavy chains H1 and H2 to form a bispecific antibody comprising the L1:H1:H2:L2 chains. Redox production methods or modified versions of the methods can be used to visualize the use of libraries/datasets of the design described herein in the production of bispecific antibodies.

In certain embodiments, cell-free protein expression systems are used to co-express polypeptides (eg, heavy and light chain polypeptides) without the use of viable cells. Instead, all components necessary to transcribe DNA into RNA and translate RNA into protein (eg, ribosomes, tRNA, enzymes, cofactors, amino acids) are provided in solution for in vitro use. In certain embodiments, in vitro expression requires: (1) a genetic template (mRNA or DNA) encoding heavy and light chain polypeptides and (2) a reaction solution containing the necessary transcriptional and translational molecular machinery. In certain embodiments, the cell extract substantially supplies components of the reaction solution, such as: RNA polymerase for mRNA transcription, ribosomes for polypeptide translation, tRNA, amino acids, enzymatic cofactors, energy sources, and cellular components essential for proper protein folding. Cell-free protein expression systems can be prepared using lysates derived from: bacterial cells, yeast cells, insect cells, plant cells, mammalian cells, human cells, or combinations thereof. Such cell lysates can provide the correct composition and ratio of enzymes and building blocks required for translation. In some embodiments, cell membranes are removed to leave only the cytoplasm and organelle components of the cell.

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

In some embodiments, cell-free expression systems are used to co-express heavy and light chain polypeptides from DNA templates (transcription and translation) or mRNA templates (translation only).

Vectors and host cells

Recombinant expression of heavy and light chains requires the construction of expression vectors containing polynucleotides encoding either heavy or light chains (eg, antibodies or fusion proteins). Once a polynucleotide encoding the heavy or light chain is obtained, a vector for production of the heavy or light chain can be produced by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods of making proteins by expressing polynucleotides containing heavy or light chains encoding nucleotide sequences. Methods 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. Accordingly, the present invention provides a replicable vector comprising a nucleotide sequence encoding a heavy or light chain operably linked to a promoter.

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

A variety of host-expression vector systems can be used to express modified heavy and light chains. Such host-expression systems represent cells capable of expressing modified heavy and light chains in situ when modified or transfected with the appropriate nucleotide coding sequence, as well as representing a vehicle from which the coding sequence of interest can be produced and then purified. . These include, but are not limited to, microorganisms such as bacteria (e.g., 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 recombinant yeast expression vectors containing modified heavy and light chain coding sequences (eg, Saccharomyces pichia); insect cell systems infected with recombinant virus expression vectors (eg, baculovirus) containing modified heavy and light chain coding sequences; Transfection with a recombinant virus expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing modified heavy and light chain coding sequences or transformation with a recombinant plasmid expression vector (eg, Ti plasmid) plant cell system; or a recombinant expression construct containing a promoter derived from a mammalian cell genome (eg, metallothionein promoter) or derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter). mammalian cell systems (e.g., COS, CHO, BHK, HEK-293, NSO, and 3T3 cells) that have In certain embodiments, bacterial cells, such as Escherichia coli, or eukaryotic cells are used for expression of modified heavy and light chains, which are recombinant antibodies or fusion protein molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) are effective expression systems for antibodies in combination with vectors such as the major intermediate early gene promoter elements from human cytomegalovirus (Foecking et al., 1986, Gene 45:101 and Cockett et al., 1990, Bio/Technology 8:2). In a specific embodiment, expression of the nucleotide sequences encoding the immunoglobulin heavy and light chains of each heterodimer is controlled by constitutive promoters, inducible promoters or tissue specific promoters.

Within mammalian host cells, a variety of viral-based expression systems can be used. When an adenovirus is used as an expression vector, modified heavy and light chains encoding sequences of interest, the coding sequences can be ligated to an adenovirus transcription/translation control complex, such as late promoter and tripartite leader sequences. Such chimeric genes can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable in an infected host and capable of expressing modified heavy and light chains (e.g., 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 initiation codon and adjacent sequences. Moreover, the initiation codon must be in the same reading frame of the coding sequence of interest to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, natural and synthetic. Expression efficiency can be increased by introduction of appropriate transcription enhancer elements, transcription terminators, etc. (eg, Bittner et al., 1987, Methods in Enzymol. 153:516-544).

Expression of the immunoglobulin heavy and light chains of the heterodimer may be controlled by any promoter or enhancer element known in the art. Promoters (eg, antibodies or fusion proteins) that can be used to control expression of genes encoding modified heavy and light chains include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78.1441-1445), the regulatory sequence of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), the tetracycline (Tet) promoter (Gossen et al. ., 1995, Proc. Nat. Acad. Sci. USA 89:5547-5551); Prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff et al, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. A plant expression vector containing the nopaline synthase promoter region (Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9: 2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); Promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and the following animal transcriptional regulatory regions (used in transgenic animals, tissue specific ): 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 region active in lymphocytes (Grosschedl et al., 1984, Cell 38:647-658; Adames et al. al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Biol. Biol. 7:1436-1444), active mouse mammary tumor virus control region in testicular, mammary, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), hepatic active albumin gene control region (Pinkert et al., 1987, Genes and Devel. 1:268-276), hepatic active alpha-fetal protein gene regulatory regions (Krumlauf et al., 1985, Mol. Biol. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); Alpha 1-antitrypsin gene regulatory region active in 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 control region active in brain oligodendrocytes (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314:283-286); nerve-specific enolase (NSE), which is active in nerve cells (Morelli et al., 1999, Gen. Virol. 80:571-83); the brain-derived neurotrophic factor (BDNF) gene regulatory region that is active in neurons (Tabuchi et al., 1998, Biochem. Biophysic. Res. Com. 253:818-823); The 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., 1999, Gen. Virol. 80: 571-83) and the active gonadotropin releasing hormone gene control region in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Additionally, host cell strains may be selected that modulate the expression of the inserted sequences, or that modify and process the gene product in a desired manner. Expression from certain promoters can be elevated in the presence of certain inducers; Thus, the expression of genetically engineered fusion proteins can be controlled. Additionally, different host cells have properties and specific mechanisms for translational and post-translational processing and modification (eg, glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be selected to ensure the desired modification and processing of the foreign protein expressed. For example, bacterial-based expression will produce an unglycosylated product, and expression in yeast will produce a glycosylated product. Eukaryotic host cells can be used that have cellular machinery for proper processing of the primary transcript (eg, glycosylation and phosphorylation) of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, HEK-293, 3T3, WI38, NSO, and in particular, neuronal cell lines such as, for example, SK. -N-AS, SK-N-FI, SK-N-DZ human glioblastoma (Sugimoto et al., 1984, J. Natl. Cancer Inst. 73: 51-57), SK-N-SH human glioblastoma ( 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 glioblastoma (Cancer 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 (Olopade et al., 1992, Cancer Res. 52: 2523-2529), C6 rat glioma cells (Benda et al., 1968, Science 161: 370-371), nerve -2a mouse glioblastoma (Proc. Natl. Acad. Sci. USA, 1970, 65: 129-136), NB41A3 mouse glioblastoma (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 elevation These 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 cell lines such as, for example, CTX TNA2 rat normal brain cortex (Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6467-6471) such as, for example, CRL7030 and Hs578Bst. Moreover, different vector/host expression systems may affect the processing response to different degrees.

Long-term, high-yield production, stable expression of recombinant proteins is often desirable. For example, cell lines that stably express the modified heavy and light chains (eg, antibodies or fusion proteins) of the present invention can be engineered. Rather than using an expression vector that contains the viral origin of replication, the host cell can be controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers. can be transformed into DNA. Following introduction of the foreign DNA, engineered cells are allowed to grow for 1-2 days in enriched medium and then switched to selective medium. Selectable markers in recombinant plasmids confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to the site of development, allowing them to be recloned and propagated into cell lines.

A number of selection systems can be used, including but not limited to: herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski Natl. can be used in Antimetabolite resistance can also be used as a basis for selecting: dhfr (confers resistance to methotrexate) (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al. , 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, (confers resistance to mycophenolic acid) (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo (confers resistance to aminoglycoside G-418) (Colberre-Garapin et al., 1981 , J. Mol. Biol. and hygro (confers resistance to hygromycin) (Santerre et al., 1984, Gene 30:147 ) gene.

Coexpression of heavy and light chains

The immunoglobulin heavy and light chains of the heterodimeric pairs described herein can be co-expressed in mammalian cells as noted above. In one embodiment, one heavy chain is co-expressed with two different light chains in the set of LCCA designs described above, wherein the heavy chain preferentially pairs with one of the two light chains. In another embodiment, the two native heavy chains are co-expressed as two native light chains, wherein each heavy chain preferentially pairs with one of the two light chains.

Testing of heterodimer pairs

As described above, at least one heterodimer of a heterodimer pair described herein may include one or more amino acid modifications to its immunoglobulin heavy chain and/or immunoglobulin light chain, whereby the heterodimer pair When the two native heavy chains and the two native light chains of are co-expressed in mammalian cells, the heavy chains of the first heterodimer preferentially pair with one of the light chains over the other. Similarly, the heavy chain of the second heterodimer preferentially pairs with the second light chain compared to the first light chain. The degree of preferential pairing can be measured, for example, by using the method described below. The affinity of each heterodimer of a heterodimer pair for its respective antigen can be tested as described below. The thermal stability of each heterodimer of a heterodimer pair can also be tested as described below.

How to Measure Preferential Pairing

LCCA

In one embodiment, preferential pairing between immunoglobulin heavy and light chains is determined by performing a light chain competition assay (LCCA). Common patent application PCT/US2013/063306, filed on October 3, 2013, describes various implementations of LCCA, which are incorporated herein by reference in their entirety for all purposes. The method allows quantitative analysis of the pairing of a heavy chain with a particular light chain in a mixture of co-expressed proteins, wherein when heavy and light chains are co-expressed, one particular immunoglobulin heavy chain is associated with one of the two immunoglobulin light chains. It can be used to measure whether or not it binds selectively. The method is described as follows: at least one heavy chain and two different light chains are co-expressed in a cell in a ratio such that the heavy chains are limiting pairing reactants; optionally isolating the secreted protein from the cell; separating the immunoglobulin light chain polypeptide that binds the heavy chain from the remainder of the secreted protein to produce a secreted protein that produces an isolated heavy chain paired fraction; detecting the amount of each different light chain in the isolated heavy chain fraction; and analyzing the relative amount of each different light chain in the isolated heavy chain fraction to determine the ability of the at least one heavy chain to selectively pair with one of the light chains.

The method provides reasonable throughput, is robust (i.e., insensitive to minor changes in operation, such as user or flow rate) and accurate. This method provides a sensitive assay capable of measuring the effect of small variations within a protein sequence. Random, protein-protein over large surface area; domain - domain; Chain-chain interactions usually require multiple mutations (swap) to introduce selectivity. The protein product does not need to be isolated and purified, which allows more efficient screening. Additional details regarding implementation of these methods are described in the Examples.

Alternative Methods for Determining Preferential Pairing

Alternative methods for detecting preferential pairing include: quantification of the relative population of heterodimers containing each light chain using LC-MS (liquid chromatography - mass spectrometry) (using their molecular weight differences to identify each individual species). Antigen activity assays can also be used to quantify the relative population of heterodimers containing each light chain, whereby the degree of binding measured (relative to a control) will be used to estimate each heterodimer population.

Additional methods such as SMCA are described in the Examples, Figures, and Tables.

thermal stability

Thermal stability of heterodimers can be determined according to methods known in the art. The melting temperature of each heterodimer is an indicator of its thermal stability. The melting point of a 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 heterodimers can be measured using circular dichroism (Murray et al. Chromatogr Sci 40:343-9).

Affinity for antigen

The binding affinity and off-rate of interaction of the heterodimer to each of these antigens can be measured by competition binding assays according to methods well known in the art. One example of a competitive binding assay is incubation of a labeled antigen (e.g., a molecule of interest (e.g., a heterodimer herein) in the presence of increasing amounts of unlabeled antigen and detection of a molecule bound to a labeled ligand. ) is a radioimmunoassay involving 3H or 125I) having a molecule of The affinity of heterodimers herein for antigen and binding off-rate can be determined from saturation data by Scatchard analysis.

Kinetic parameters of the heterodimers described herein can also be measured using surface plasmon resonance (SPR) (eg, BIAcore kinetics analysis) known in the art. For a review of SPR-based techniques, see Mullet et al., 2000, Methods 22: 77-91; Dong et al., 2002, Review in Mol. Biotech., 82: 303-23; Fivash et al., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al., 2000, Current Opinion in Biotechnology 11: 54-61. Additionally, any of the SPR devices and SPR-based methods for measuring protein-protein interactions described in U.S. Patent Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; 6,268,125 is contemplated as being in the method of the present invention. FACS can also be used to measure affinity, as is known in the art.

Generation of bispecific antibodies predetermined Mab1 and Mab2 using a library of bispecific antibody mutation design sets

In one embodiment, described herein is a set of bispecific antibody mutation designs aimed at selectively forming the disclosed bispecific antibodies from two canonical antibodies Mab1 and Mab2 comprising antigen-binding fragments Fab1 and Fab2, respectively. . The design set consists of mobilization mutations corresponding to each of Fab1, Fab2 and Fc. In one embodiment, the design set library is represented by a design set included in Table 5, Table 12, or any of Tables 15-17. Mutations are introduced at the interface of the light and heavy chains of Fab1 to achieve selective pairing between the two obligate chains in the presence of competing light and heavy chains of Fab2. Selective pairing is achieved by introducing good complementary mutations in the two obligate light and heavy chains based on steric, hydrophobic, and electrostatic complementarity between certain frequent framework residues at the interface. In each design set, a selective pairing mutation is also introduced at the interface of the light and heavy chains of Fab2 to achieve selective pairing between the two obligate chains in the presence of competing light and heavy chains of Fab1. The mutation aims to reduce the mispairing of the light chain from Fab1 with the heavy chain from Fab2 and vice versa. Mutations are introduced at the Fc interface to achieve selective pairing of the heavy chains, forming an asymmetric antibody molecule comprising two different heavy chains.

Engineering at specific interfacial residue positions of the light and heavy chains of an antibody can often lead to deleterious effects, such as a decrease in the antibody's antigen binding affinity, stability, solubility, aggregation propensity, and the like. A number of relevant properties may be affected, such as kon and koff ratios, melting temperature (Tm), acid, base, oxidation, freeze/thaw, stability or stress conditions such as agitation, pressure, and the like. This is often influenced by the complementarity determining regions (CDRs) of the antibody of interest. As the CDRs of different antibodies are generally not identical, the impact of designing a set of mutations on the properties described above may not be the same across the entire antibody. Given any two available antibodies, Mab1 and Mab2, methods for forming bispecific antibodies with the stated purity compared to other contaminants containing incorrectly paired antibody-like structures are presented herein. The light and heavy chains of Mab1 and Mab2 are co-expressed after introduction of mobilization mutations in each of the mutant design sets, and the expressed antibody products are analytically screened to determine the purity of the desired bispecific antibody by comparison with other Mab-like species expressed in the protein. estimate In some embodiments, the analytical screening procedure can be based on LC-MS techniques. In some embodiments, analytical screening procedures can be based on charge-based separations, such as capillary isoelectric focusing (cIEF) techniques or chromatographic techniques. An example of a screening technique is presented in Example 9 based on the SMCA procedure. In some embodiments, the stated purity of the bispecific antibody is defined as greater than 70% of the total species of Mab obtained within the expressed protein product. In some embodiments, the stated purity of the bispecific antibody is defined as greater than 90% of the total number of Mab species obtained within the expressed protein product. The procedure for the construction and selection of bispecific Mab design sets for Mab1 and Mab2 is shown schematically in FIG. 12 .

pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising a heterodimer or heterodimer pair described herein. Such compositions include a therapeutically effective amount of the heterodimer or pair of heterodimers and a pharmaceutically acceptable carrier. In certain embodiments, the term “pharmaceutically acceptable” is approved by a regulatory agency of a federal or state government or approved by a U.S. regulatory agency. pharmacopoeias or other generally recognized pharmacopeias for use in animals, more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, such as oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. 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, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water , ethanol, etc. are included. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The composition may be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may include excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Suitable examples of pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with an appropriate amount of carrier to provide a form for proper administration to the patient. The formulation should suit the mode of administration.

In certain embodiments, a composition comprising a heterodimer or pair of heterodimers is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to relieve pain at the site of the injection, such as lignocaine. Generally, these ingredients are presented separately or together in unit dosage form, eg, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, eg, an ampoule or bag indicating the amount of active ingredient. mixed If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline may 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 those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like, and those formed with cations such as sodium, potassium, ammonium, calcium, ferric hydroxide isopropylamine , triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The amount of a composition described herein that will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also be based on the route of administration and severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances. Effective doses are extrapolated from dose-response curves derived from in vitro or animal model evaluation systems.

Uses of heterodimer pairs

As described above, the heterodimer pairs described herein include a first heterodimer and a second heterodimer, wherein the immunoglobulin heavy chain and/or immunoglobulin light chain of each heterodimer binds to a known molecule. It may contain one or more modifications from a known therapeutic antibody, or from a known therapeutic antibody. Thus, heterodimers containing modifications to these antibodies can be used for the treatment or prevention of known therapeutic antibodies or homologous diseases, disorders, or infections for which known antibodies can be used.

In another embodiment, the heterodimer pairs described herein may also be advantageously used in combination with other therapeutic agents known in the art for the treatment or prevention of cancer, autoimmune disease, inflammatory disorder or infectious disease. there is. In certain embodiments, heterodimeric pairs described herein may be used in combination with monoclonal or chimeric antibodies, lymphokines or hematopoietic growth factors (such as, for example, IL-2, IL-3 and IL-7). , which act, for example, to increase the number or activity of effector cells that interact with the molecule and increase the immune response. Heterodimer pairs described herein may also be advantageously used in combination with one or more drugs to treat a disease, disorder, or infection, such as, for example, an anti-cancer agent, an anti-inflammatory agent, or an anti-viral agent. .

kit

The invention further provides kits comprising one or more heterodimer pairs. The individual components of the kit will be packaged in and associated with such containers in separate containers, notices reflecting approval by the manufacturing, using, or marketing authorities by governmental authorities regulating the manufacture, use, or sale of pharmaceuticals or biological products. It may be a notice in a prescribed form. The kit may optionally contain instructions or instructions outlining methods of use or dosing regimens for the heterodimer pair.

Where one or more components of the kit are provided as a solution, eg an aqueous solution, or a sterile aqueous solution, the container means itself an inhaler, syringe, pipette from which the solution can be administered or applied to a subject and mixed with the other components of the kit. , eye drops, or other such similar devices.

Components of the kit may also be provided in dried or lyophilized form, and the kit may additionally contain a suitable solvent for reconstitution of the lyophilized components. Regardless of the number or type of containers, the kits of the present invention may also include a device to assist in administering the composition to a patient. Such a device may be an inhaler, nasal spray device, syringe, pipette, forceps, measuring spoon, eyedropper, or similar medically approved delivery vehicle.

computer running

In one implementation, a computer includes at least one processor coupled to a chipset. Also associated with the chipset are memory, storage devices, keyboards, graphics adapters, pointing devices, and network adapters. A display is coupled with a graphics adapter. In one implementation, the functionality of the chipset is provided by a memory controller hub and an I/O controller hub. In another implementation, the memory is coupled directly with the processor instead of with the chipset.

A storage device is any device capable of holding data, such as a hard drive, compact disc readable memory (CD-ROM), DVD, or solid-state memory device. Memory holds instructions and data used by the processor. The pointing device may be a mouse, track ball or other type of pointing device and is used in combination with a keyboard to enter data into the computer system. A graphics adapter presents images and other information on a display. A network adapter couples a computer system to a local or wide area network.

As is known herein, a computer may have different and/or other components than those previously described. Additionally, the computer system may be free of certain components. Moreover, the storage device may be local and/or remote from the computer (eg, a storage area network (SAN)).

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

It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications thereof are suggested to those skilled in the art and are included within the spirit and scope of the present application and the scope of the appended claims. do.

Example

Below are examples of specific implementations for practicing 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 errors and deviations should, of course, be tolerated.

The practice of the present invention will employ conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, unless otherwise specified, within the skill of the art. This technique is fully explained in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

Example 1: Molecular Modeling and Computational Guided Processing of the Fab Interface

Structural and computational molecular modeling-guided approaches have been used to generate libraries of heavy and light chain mutation designs that can be screened in the context of other antibodies (Abs) or fractions thereof to identify mutations exhibiting specificity of interest in an antibody of interest. . The design strategy for processing preferential heavy chain (H)-light chain (L) chain pairing involved the first identification of a representative Fab (ie D3H44).

As pointed out in Table 1, the key criteria for these Fabs are that they are human/humanized, have the commonly used VH and VL subgroups, and contain minimal framework region mutations. In addition, structural considerations should ensure that the VH:VL interdomain angle is close to the average observed for the antibody. After selection of Fab D3H44 , in silico analysis of the Fab interface was performed to identify and identify residues important for interaction between the heavy and light chains using a two-pronged approach.

The first approach involved in a global analysis of sequence conservation across the Fab variable and constant interfaces was carried out through sequence and structural alignment of known antibodies. An alignment of the constant and variable domain sequences from the various antibody subgroups is shown in FIG. 1 . 1A depicts the alignment of representative human VH germline subgroups. 1B depicts the alignment of representative human kappa VL germline subgroups. 1C depicts the alignment of representative human lambda VL germline subgroups. 1D depicts an alignment of human CH1 allelic sequences. Figure 1E depicts an alignment of human kappa and lambda allelic sequences. A second approach involved analysis of the D3H44 crystal structure interface using a number of molecular modeling tools as shown in FIG. 2 (eg ResidueContacts ^™ ). These analyzes resulted in the identification of a list of frequent sites for processing of preferential HL pairing. Frequent locations determined from this analysis are listed in Table 2. These positions and amino acids (with the exception of a few located in the CDR3 loops) are primarily framework residues and are also largely conserved in the lambda L chain. Amino acids in the parent D3H44 sequence with Kabat numbering are provided in Tables 3a-3b.

Next, potential mutations at the frequent positions of interest as well as at positions adjacent to the frequent positions in the 3D crystal structure were simulated and identified through in silico mutagenesis and packing/modeling using Zymepack ^™ . Zymepack ^™ is a software suite that, given an input structure and a set of mutations, will modify the type of residues in the input structure according to the supplied mutations, and will generate a new structure that approximates the physical structure of the mutant protein. Additionally, Zymepack evaluates the properties of mutant proteins by computing a variety of quantitative matrices. These matrices include assessment of steric and electrostatic complementarity, which can correlate with stability, binding affinity, or heterodimer specificity of the mutant protein.

Figure 3 shows a subset of frequent positions at the heavy and light chain interfaces in the variable domains and mutations can be introduced at these interface positions to promote selective pairing of obligate chains while disfavoring the formation of imprecise chain pairs. prove that there is Using computational methods including Zymepack ^™ , steric complementarity was modeled and calculated based on energy factors such as van der Waals packing, cavitation effects and close contact of hydrophobic groups. Similarly, electrostatic interaction energies were modeled and evaluated based on Coulombic interactions between charges, hydrogen bonding, and desolvation effects. All preferred heavy and light chain pair models such as H1:L1 (or H2:L2) and imprecise pair models such as H1:L2 (and H2:L1) obtained by introduction of the mutations of interest are used to compute relative conformational and electrostatic scores. has been imitated This allowed determination of whether a particular set of mutations led to a preferential energy, ie greater steric and/or electrostatic complementarity, for the preferred (obligate) heavy chain-light chain pair over the incorrect (non-obligate) pair. The computed steric and electrostatic energies are components of the free energy associated with light and heavy chain pairing. Thus, greater steric and electrostatic complementarity indicates a greater free energy change associated with pairing of obligate pairs compared to pairing of non-obligate pairs. Greater steric or electrostatic complementarity results in preferential (selective) pairing of obligate heavy and light chains over non-obligate pairs.

Example 2: Design selection and description

The approach described in Example 1 was used to design heavy-light chain heterodimer pairs ( ie H1-L1 and H2-L2) that exhibit selective or preferential pairing. Heterodimers are designed in pairs, referred to as “designs” or “design sets,” and contain sets of substitutions on the H1, L1, H2, and L2 chains that promote preferential pairing (Table 5). A set of designs were initially tested as "LCCA designs" (Table 4) in which one heavy chain was co-expressed with two light chains to assess relative pairing. Amino acid substitutions are identified with reference to Tables 3a, 3b using the Kabat numbering system.

The design library listed in Table 30 from International Patent Application No. PCT/CA2013/050914 was used as a starting point to identify the LCCA designs shown in Table 4 and part of the set of designs shown in Table 5. Some of the designs in Tables 4 and 5 are new independent designs. The core design is shown in Table 6 along with associated unique identifiers. Most designs span only the constant region, and a few of the designs also incorporate variants in the variable region. These designs are proposed to further drive pairing specificity while also favoring portability to other antibody systems.

For the directed design, a library of designs set forth in Table 30 from International Patent Application No. PCT/CA2013/050914 was used as measured by the strength of pairing specificity, the effect of antigen binding, and differential scanning calorimetry (DSC). Designs, ranked based on stability and clustered by structural similarity, were used as starting points. The designs were then combined (Examples in Reference Table 7) and/or optimized (Examples in Reference Tables 8 and 9) to obtain derived designs. For combinations, at least one of the designs exhibited a high pairing specificity while the other design(s) exhibited a range of preferred pairing specificities. All designs selected for combination and/or optimization showed no/minimal effect on antigen binding and no/minimal effect on melting temperature (Tm).

Independent designs are available alone (classified as independent under Design Type column, Table 5) and in combination with derived designs (classified as independent/combination under Design Type column, Table 5; see also Examples in Table 10) as well as derived designs. has been tested

The design was packed on a molecular model of D3H44 and the matrix calculated (as described in Example 1). The top design was then selected based on risk (possible effects on immunogenicity as well as stability) and impact (taking into account the proposed strength of the driving pairing specificity). These top 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. The D3H44 Fab light chain (AJ308087.1) and heavy chain (AJ308086.1) sequences were selected from GenBank (Table 3c), gene synthesized and codon optimized for mammalian expression. The light chain vector insert, consisting of 5′-EcoRI cut region - HLA-A signal peptide - HA or FLAG tag - light chain Ig clone - 'TGA stop' - BamH1 cut region-3', was ligated into the pTT5 vector (Durocher Y et al. al., Nucl. Acids Res. 2002;30, No.2 e9). The obtained vector + insert was sequenced to confirm the correct reading frame and sequence of the coding DNA. Similarly, the heavy chain vector insert, consisting of 5'-EcoR1 cut region - HLA-A signal peptide - heavy chain clone (terminated at T238; see Table 3a) - ABD ₂ -His ₆ tag - TGA stop - BamH1 cut region-3' ligated into the pTT5 vector (ABD; albumin binding domain). The resulting vector + insert was also sequenced to confirm the correct reading frame and sequence of the coding DNA. For the design set, various Fab D3H44 constructs containing amino acid substitutions were generated either by gene synthesis or by site-directed mutagenesis (Braman J, Papworth C & Greener A., Methods Mol. Biol. (1996) 57:31 -44).

The heavy and light chains were tagged at the C- and N-terminus, respectively, to facilitate evaluation of preferential pairing via a competition assay-SPR screen. The ABD ₂ -His ₆ heavy chain tag specifically allowed HL complexes to be captured on the anti-his tag SPR chip surface, while the FLAG and HA light chain tags allowed the relative L1 and L2 populations to be quantified.

Example 4: Assessment of preferential pairing of Fab heterodimers comprising constant domain modifications or combinations of constant and variable domain modifications in D3H44 IgG light and/or heavy chains.

Constructs encoding D3H44 IgG heavy and light chains in Fab format containing amino acid modifications according to the LCCA design set in Table 12 were prepared as described in Example 3. The ability to pair preferentially to construct the desired H1-L1 heterodimer in the context of the LCCA design set (H1, L1, L2) was measured using a light chain competition assay (LCCA).

LCCA quantifies the relative pairing of one heavy chain to at least two native light chains and can be summarized as follows. One D3H44 heavy chain Fab construct was co-expressed with two native D3H44 light chain Fab constructs and the relative light chain pairing specificities (e.g. H1-L1:H1-L2) were evaluated in a competition assay-SPR, performed in duplicate. determined from the screen. By reducing the amount of L1 (designed for preferential pairing with the H chain) compared to L2 (e.g. L1:L2 = 1:3, by weight), while limiting the heavy chain (i.e. 1:3 of H1 : L1 + L2), the LCCA screen ratio was skewed to one side to confirm the strong driver. The amount of each heterodimer ( i.e. , H1-L1 and H1-L2) formed was determined by binding the heavy chain to an SPR chip via his-tag pull-down, and then using antibodies specific for these tags to determine each light chain tag ( HA or FLAG) was detected. Selected heterodimer hits were then confirmed via validation of a light chain competition assay whereby the L1:L2 DNA ratio varied by 1:3 and 1:9 during transfection, while maintaining limited amounts of heavy chain. Note also that the light chain tag (HA or FLAG) does not affect LCCA pairing in the D3H44 system (Example 10 from reference International Patent Application No. PCT/CA2013/050914). A schematic representing the design of the assay is shown in FIG. 4 . Figure 5 depicts how heavy and light chains are tagged and how preferential pairing is assessed. Experimental details of LCCA are provided below.

Transfection method

An LCCA design comprising one heavy chain and two light chain constructs, prepared as described in Example 3, was transfected into CHO-3E7 cells as follows. CHO-3E7 cells at a density of 1.7 - 2 x 10 ⁶ cells/ml were cultured at 37°C in FreeStyle ^™ F17 medium (Invitrogen cat# A-1383501) supplemented with 4 mM glutamine and 0.1% KoliphorP188 (Sigma #K4894). . A total volume of 2 ml was transfected with a total of 2 μg DNA using PEI-Pro (Polyplus transfection # 115-375) at a DNA:PEI ratio of 1:2.5. 24 hours after the addition of the DNA-PEI mixture, the cells were shifted to 32°C. Supernatants were tested for expression on day 7 by non-reducing SDS-PAGE analysis followed by Coomassie blue staining to visualize bands. H:L ratios are as indicated in Table 11.

Competition assay SPR method

The degree of preferential D3H44 light chain pairing to the D3H44 heavy chain in the LCCA design was assessed using SPR-based reading of unique epitope tags located at the N-terminus of each light chain.

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

SPR biosensor assay. All surface plasmon resonance assays were performed at a temperature of 25°C using a BioRad ProteOn XPR36 instrument (Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON) and PBST working buffer (PBS Teknova Inc with 0.05% Tween20). An anti-penta His capture surface was created using a GLM sensorchip activated by a 1:5 dilution of standard BioRad sNHS/EDC solution injected for 140 s at 100 μL/min in analyte (horizontal) orientation. , a 25 μg/mL solution of anti-penta His antibody (Qiagen Inc.) in 10 mM NaOAc pH 4.5 in the analyte (vertical) orientation at a flow rate of 25 μL/min until approximately 3000 resonance units (RUs) were immobilized. Residual active groups were quenched with a 140 s injection of 1M ethanolamine at 100 μL/min in the analyte orientation, which also confirms that mock-activated interspots were created for the blank reference.

Screening of heterodimers for binding to anti-FLAG (Sigma Inc.) and anti-HA (Roche Inc.) monoclonal antibodies occurred in two steps: Indirect capture followed by anti-FLAG and anti-HA injections in analyte orientation. First, 1 injection of PBST for 30 s at 100 μL/min with the ligand orientation was used to stabilize the baseline. For each heterodimer capture, unpurified heterodimers in cell-culture medium were diluted to 4% in PBST. 1 to 5 heterodimers or controls (i.e. controls containing 100% HA-light chain or 100% FLAG-light chain) were simultaneously injected into individual ligand channels at a flow rate of 25 μL/min for 240 s, resulting in anti-penta His surface This resulted in approximately 300-400 RUs of saturated heterodimer entrapment on the phase. The first ligand channel was emptied to serve as a blank control if necessary. This heterodimer capture step was immediately followed by two buffer injections in the analyte orientation to stabilize the baseline, followed by 50 μL of 5 nM anti-FLAG and 5 nM anti-HA each for 180 s dissociation phase for 120 s. Repeated scanning at /min resulted in a set of combinations of buffer references and sensorgrams for each entrapped heterodimer. Tissue factor (TF) antigen to which the heterodimer binds was also injected across the last remaining analyte channel as an active control. The heterodimer was regenerated with 18 s pulses of 0.85% phosphoric acid for 18 s at 100 μL/min to prepare the anti-penta His surface for the next injection cycle. The sensorgrams were aligned and double-referenced using buffer background scan and interspot, and the resulting sensorgrams were analyzed using ProteOn manager software v3.0.

result

LCCA results are shown in Tables 12, 13a and 14a. In Tables 13 and 14, as the unique identifier for the two component LCCA can be of either orientation ((set#H1L1L2-set#H2L2L1) or (set#H2L2L1-set#H1L1L2)), “unique identifier” cannot correspond correctly to Table 5. The evaluation of preferential pairing for each LCCA design is shown in the last three columns of Table 12. The same data is also included in the context of design pairs in Tables 13a and 14a in columns 5, 6, and 8, or 10, 11 and 13. Each unique set of H1, L1 and L2 mutations (LCCA design) was assigned a unique number, or 'set #' (eg 9567 or 9087). When data appears in the H1 L1 H2 L2 format (Fab pair format or design set), such design set results in a 'unique identifier' consisting of a set number for two component LCCAs (e.g. 9567-9087). is indicated by Note that the majority of LCCA experiments were performed on constructs containing interchain Fab disulfide bond(s) located in the constant domains (H/C233-L/C214, Kabat numbering). Within Tables 13(a and b) and 14(a and b), for the purpose of highlighting specific designs for preferential pairing, two complementary LCCA sets (H1, L1, L2 and H2, L2, L1) is represented in Fab pair format. The presence of tags (L chain: HA and FLAG and H chain: ABD ₂ -His ₆ ) did not affect the expected neutral pairing of -50%:50% for D3H44 WT.

In the table, reported LCCA data are median in ratio format (H1-L1:H1-L2 and H2-L2:H2-L1) normalized to an L1:L2 DNA ratio of 1:1. Moreover, the LCCA data were normalized to 100%, as the total amount of L1 and L2 was observed for some variants that were significantly 100% different. It is believed that this difference in total light chain percentage is due in part to the occurrence of variable non-specific binding during initial heterodimer capture on the SPR chip. As LCCA experiments were performed at 2 different L1:L2 DNA ratios (L1:L2 1:3 and 1:9, respectively), all LCCA normalized ratios are listed in the table. As the experimental data obtained did not meet the inclusion body criterion (e.g. Fab capture on the SPR chip was less than 100, or the total amount of LCCA in L1 and L2 was of interest outside the range of 60 to 140), the LCCA data could be attributed to some LCCA Note that the experiment was not reported.

Table 12 lists all LCCA designs (530) for which data were obtained. Except for 530 LCCA designs, 490 (92.5%) of these LCCA designs are at least 60% correct (at normalized L1:L2 DNA ratios of 1:1), taking into account all L1:L2 DNA ratios of 1:3 and 1:9. had twin formation. The remaining LCCA designs included predominantly neutral (32/530 or 6.0%) LCCA designs as well as a small fraction that gave inconsistent (8/530 or 1.5%) results. The designs shown in Table 12 are predominantly electrostatic (based on specificity drivers using hydrogen bonds or charge-charge interactions) and some designs also included steric complementarity and/or interchain covalent disulfide bonds. Some designs also included mutations for the formation of novel disulfide bonds in the absence of natural interchain disulfide bonds (formed by H/C233-L/C214).

Tables 13 (a and b) and 14 (a and b) list 447 designs for which LCCA data represent all heterodimers in the design set. Tables 13a and 14a show that the in silico design approach described in Example 1 leads to the achievement of preferential pairing of H1-L1 to H1-L2 and that of H2-L2 to H2-L1 over a diverse set of designs and variations thereof. prove that you did

Table 13 (a and b) shows the average LCCA performance of paired:mispaired Fab heterodimers of at least 86:14 (L1:L2 ratio 1 for H1-L1:H1-L2 and H2-L2:H2-L1). Table 14 (a and b) lists designs with an average LCCA performance of paired:error paired Fab heterodimers of less than 86:14, whereas Table 14 (a and b) lists designs with average LCCA performance of paired:error paired Fab heterodimers of less than 86:14 do. The performance of each LCCA was normalized to 100% as well as to an L1:L2 DNA ratio of 1:1 (as described in these examples above), and all scalar values ((ln(r1/f1) or ln(r2/f2) ) (where r1 and r2 correspond to the median values of H1L1:H1L2 and H2L2:H2L1 in empirical ratios, respectively, and f1 and f2 correspond to the respective empirical ratios) as well as pairing versus mispairing Fab heterogeneity Each design also has an associated average LCCA performance scalar value (0.5(ln( 447 Except for the Mab designs, 354 (79.2%) exhibited at least an average LCCA performance of 86:14 (Tables 13 a and b) The designs in Table 13 (a and b) were further characterized into 13 clusters based on similarity of designs Designs within each cluster were ordered from highest to lowest mean LCCA performance scalar value.

In addition, the LCCA data in Table 13a is also graphically represented in FIG. 7 . Figure 7 depicts a box plot showing average LCCA performance values of at least 86:14 paired:mispaired Fab heterodimers for each cluster. The bottom of each box represents the first quartile (Q1), which is the median average LCCA performance value between the minimum and median values, whereby values below the ^{1st quartile} represent the lowest 25% of the data. The horizontal bar inside the box represents the second quartile, which is the median average LCCA performance value for the cluster. The top of each box represents the 3rd quartile (Q3), which is the median mean LCCA performance value between the highest and median values, whereby values above the ^3rd quartile represent the highest 25% of the data. The interquartile area is the difference between Q3 and Q1. Whiskers extending vertically in all orientations indicate data ranges for values that are within Q1 - (1.5 * IQR) or Q3 + (1.5 * IQR). The horizontal bars covering the whiskers represent the maximum and minimum values within the range. Data and whiskers outside the box plot are light outliers labeled with dots (different from Q1 or Q3 by 1.5*IQR to 3*IQR) and plus points (different from Q1 or Q3 by more than 3*IQR). (plus) are identified as outliers, with significant outliers labeled.

Example 5: Scale-up for biophysical characterization

As indicated in the set of unique identifiers (Table 5), correctly paired heterodimers were scaled up (typically to 20 ml) and purified as follows to test for thermal stability and antigen binding. The heavy and light chains of each heterodimer were expressed in 20 ml cultures of CHO-3E7 cells. At a density of 1.7 - 2 x 10 ⁶ cells/ml, CHO-3E7 cells were cultured at 37°C in FreeStyle ^™ F17 medium (Invitrogen cat# A-1383501) supplemented with 4 mM glutamine and 0.1% Koliphor P188 (Sigma #K4894). has been cultured A total volume of 20 ml was transfected with a total of 20 μg DNA using PEI-Pro (Polyplus cat# 115-375) at a DNA:PEI ratio of 1:2.5. 24 hours after the addition of the DNA-PEI mixture, the cells were shifted to 32°C.

Cells were centrifuged 7 days after transfection and heterodimers were purified from the supernatant by high-throughput nickel affinity chromatography purification as follows. Supernatants were diluted with cell culture supernatants to 20 - 25% in equilibration buffer (Dulbecco's Phosphate Buffered Saline (DPBS) without calcium, magnesium, and phenol red (HyClone ^™ # SH30028.02)) and then HisPur® for 12 hours. It was incubated while mixing with Ni-NTA resin (Thermo Scientific # PI-88222) and pre-equilibrated with equilibration buffer. The resin was then collected by centrifugation, transferred to a 96 well-melted plate, washed three times with equilibration buffer and eluted using HIS-Select® Elution Buffer (Sigma-Aldrich # H5413).

After purification, heterodimer expression was assessed by non-reducing high-throughput protein express assay using Caliper Labchip GXII (Perkin Elmer #760499). The procedure was performed according to the HT Protein Express LabChip User Guide Version 2 LabChip GXII User Manual with the following modifications. Heterodimer samples at 2 μl or 5 μl (concentration range 5-2000 ng/μl) were added to separate wells in a 96 well plate (BioRad # HSP9601) with 7 μl HT Protein Express Sample Buffer (Perkin Elmer # 760328) made it Heterodimer samples were then denatured at 70°C for 15 minutes. The LabChip instrument was run using the HT Protein Express Chip (Perkin Elmer #760499) and Ab-200 assay settings. After use, the chips were rinsed 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 (DSF) was used as a high-throughput method to screen all correctly paired heterodimers compared to those of wild-type, unmodified heavy-light chain pairs. Heterodimers were 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 in DPBS (HyClone Cat # SH30028.02). For the majority of samples, a working stock of Cipro Orange Gel Stain (Life Technologies Cat # S-6650) was prepared by diluting 4 μL of Cipro Orange Gel Stain in 2 ml DPBS. DSF samples were prepared by adding 14 μL of 0.5 mg/mL protein to 60 μL of diluted cipro orange gel stain working stock. However, for proteins with less than 0.5 mg/mL, each DSF sample adds 14 μL of undiluted protein to a 60 μL working stock of Sypro Orange dye (diluted 1:1500 in DPBS). made by doing DSF analysis was then performed in duplicate on 20 μl aliquots using a Rotor-Gene 6000 qPCR machine (QiaGen Inc). Each sample was scanned from 30°C to 94°C using a 30 second wait time at start and 1°C interval with 10 second equilibration between each step. An excitation filter of 470 nM and an emission filter of 610 nM in increments of 9 were used. Data were analyzed with Rotor-Gene 6000 software using the maximum value from the first derivative of the denaturation curve as Tm. The remaining DSF samples were similarly prepared and analyzed with the following protocol modifications not altering the measured Tm values: 1) working stocks were prepared by diluting 1 mL of Cipro Orange gel stain in 2 ul DPBS, 2) 30 μl Aliquots were analyzed and 3) increments of 10 were used.

DSF results are shown in Tables 12, 13b and 14b. The thermal stability (DSF value and DSF value compared to wild type) of the H1:L1 Fab in the context of the LCCA design is shown in columns 3 and 4 of Table 12. The same DSF values are also included in the context of design pairs in Tables 13b and 14b, columns 7 and 8. For each Fab heterodimer subjected to repetition, the reported Tm value is the median value. A comparison of the Fab heterodimer Tm values to the Tm values of the wild-type Fab heterodimer (the wild-type Fab construct containing the HA tag, with a median Tm of 81.0 °C) is reported in the H1L1_dTm_dsf column. Note that for several Fab heterodimers lacking native interchain disulfide (between H chain C233 and L chain C214), no H1L1_dTm_dsf value was determined as the corresponding wild-type Fab lacking native interchain disulfide was not evaluated. Also, due to the quality of each experiment (e.g., low yield, low intensity, partially occluded peaks, and variability between repetitions of Fab heterodimers above 1 °C), some Fab heterodimers have reported Tm values (Fab 17/230 or 7.4% of heterodimers). For some of these Fab heterodimers, estimated Tm values are reported instead, corresponding to Tm values from similar Fab heterodimers that differ only in the presence/absence or identity of the attached L chain tag (HA or FLAG). For the estimated Tm values, the corresponding wild-type Tm value (81.2° C.) is the median value obtained from all wild-type Fab heterodimer constructs (i.e. Fab constructs containing an HA tag or a FLAG tag). HA or FLAG tags do not significantly affect the Tm values of wild-type Fab heterodimers. Overall, Fab heterodimers showed similar Tm values compared to WT. Among the Fab heterodimers containing native interchain disulfides and also for which DSF data are available, 93% (195/209) of the Fab heterodimers exhibited a loss of 3 °C or less relative to WT. Moreover, the most affected Fab heterodimer showed a loss of 6.5 °C relative to WT. Table 12 lists LCCA designs in decreasing Tm rank order.

Moreover, a 13 amino acid substitution was identified that generally improved the stability of Fab heterodimers (see Table 34). Stabilizing mutations were identified following comparison of Fab heterodimers with stabilizing mutations versus similar Fab heterodimers that differ without 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, stabilizing mutations increased stability by 0.4°C to 2.1°C. The 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, 0.4°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, respectively. The light chain stabilizing mutations Q124E, Q124R, Q160F, S176L, and T180E increased stability by 0.4°C to 0.5°C, 0.8°C to 0.9°C, 0.6°C, 0.4°C to 1.0°C, and 0.5°C, respectively.

Example 7: Determination of antigen affinity of Fab heterodimers.

The ability of Fab heterodimers to bind tissue factor was evaluated to determine whether amino acid substitutions had any effect on the heterodimer's ability to bind antigen. The affinity of each Fab heterodimer for tissue factors was determined by SPR as follows.

SPR supply. The GLC sensorchip, 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 working buffer with 0.05% Tween20 (PBST) is from Teknoca Inc. (Hollister, CA).

Fab heterodimer placement. Purified Fab heterodimers were tested in 3 batches, A, B, and C. Batches A and B were stored at 4°C for approximately 1 month prior to performing the SPR assay, whereas Fab heterodimers purified from batch C were stored at 4°C for approximately 2 months prior to performance of the SPR assay. Fab heterodimers from batch C are indicated by 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 at a temperature of 25°C. An anti-penta His capture surface was created using a GLC sensorchip activated by a 1:5 dilution of standard BioRad sNHS/EDC solution injected for 140 s at 100 μL/min in analyte (horizontal) orientation. Immediately after activation, a 25 μg/mL solution of anti-penta His antibody (Qiagen Inc.) in 10 mM NaOAc pH 4.5 was applied to the analyte (vertically) at a flow rate of 25 μL/min until approximately 3000 resonance units (RUs) were immobilized. injected in the same orientation. Residual active groups were quenched with a 140 s injection of 1M ethanolamine at 100 μL/min in the analyte orientation, which also confirmed that mock-activated interspots were created for the blank reference.

Screening of Fab heterodimers for binding to TF antigen occurred in two steps: indirect capture of Fab heterodimers on the surface of an anti-penta-His antibody in ligand orientation followed by 5 concentrations of double reference in analyte orientation. Simultaneous injection of purified antigen and 1 buffer background. First, the baseline was stabilized with 1 buffer injection for 30 s at 100 uL/min in the ligand orientation. At a concentration of 3.4 μg/ml in PBST, 1 to 5 variants or controls were simultaneously injected in individual ligand channels for 240 s at a flow rate of 25 μL/min. This resulted in an average entrapment of approximately 1000 RU on the anti-penta His surface for batches A and B, and an average entrapment of approximately 600 RU on the anti-penta His surface for batch C. The first ligand channel was emptied for use as a blank control if necessary. This capture step was followed immediately by injecting 2 buffers at 100 μL/min for 30 s each in the next analyte orientation to stabilize the baseline, followed by 60 nM, 20 nM, 6.7 nM, 2.2 nM and 0.74 nM antigen with buffer blank. (TF) were simultaneously injected at 50 μL/min for 120 s with a 600 s dissociation phase. The captured antibody surface was regenerated by two 18 s pulses of 0.85% phosphoric acid for 18 s at 100 μL/min to prepare for the next injection cycle. The sensorgrams were aligned and double-referenced using Buffer Blank Scan and Interspot, and the resulting sensorgrams were analyzed using ProteOn manager software v3.1. The double-referenced sensorgram was fitted to a 1:1 binding model. Rmax values for each antigen were normalized to the level of antibody capture for each variant and compared to the 100% control.

Antigen affinity (KD) values for Fab heterodimer samples are reported in Tables 12, 13b and 14b. The KD values (KD, range of KD values, and change in median KD value compared to wild type) of the H1:L1 Fab in the context of the LCCA design are shown in Table 12, columns 5, 6, and 7, respectively. The same KD values are also shown in Tables 13b and 14b, columns 3 (KD of H1-L1 Fab heterodimers), 4 (KD change of H1-L1 Fab heterodimers compared to wild type), 5 (H2-L2 Fab heterodimers). KD of the heterodimer), and 6 (change in KD of the H2-L2 Fab heterodimer compared to wild type) in the context of design pairs. KD values were determined only for Fab heterodimer samples showing at least 100 RU of Fab heterodimer entrapment. The reference wild-type KD (0.157 nM) reflects the median value of wild-type Fab heterodimers in which the light chain contains a FLAG tag. Wild-type Fab heterodimers (containing FLAG or HA tags) showed similar KD values, whereby a 2.6-fold difference was observed between the maximum and minimum values. In Tables 12, 13b and 14b, the KD difference for the wild-type antigen binding affinity is shown using the calculation - (log(KD) design - log(KD)wt), whereby positive values indicate lower KD values, whereas Negative values in a indicate an increased KD value of the Fab heterodimer compared to the wild-type binding affinity for the antigen. Note that some Fab heterodimers lack the measured KD values. In some of these cases, Fab heterodimers were evaluated but SPR experiments showed low Fab heterodimer entrapment (i.e. less than 100 RU) and therefore precise determination of KD values was not possible. For Fab heterodimers that show similarity to other Fab heterodimers (i.e. differ only in the presence/absence or identity of an attached L chain tag (HA or FLAG)), the estimated KD values are (in Tables 12, 13b and 14b As noted), they correspond to KD values from similar Fab heterodimers. The corresponding estimated wild-type KD value (0.15 nM) was the median from all wild-type Fab heterodimer constructs (i.e. Fab constructs containing an HA tag or a FLAG tag). Overall, the results indicate that (from a design point of view) correctly paired heterodimers exhibit wild-type-like binding affinities (within approximately 2.3 times the reference wild-type affinity) for the antigen.

Example 8. Ultra Performance Liquid Chromatography Size Exclusion Chromatography (UPLC-SEC) profiles of wild-type tagged D3H44 heterodimers and preferentially paired heterodimers.

Wild-type D3H44 heterodimers (one heavy chain and one light chain) with a C-terminal ABD2-His ₆ tag on the heavy chain and an N-terminal tag on the light chain (FLAG in one construct and HA in another construct) are in the art. It was expressed and purified according to methods similar to those known in and described in Example 5. Preferentially or correctly paired heterodimers were individually scaled up and purified via His tag affinity purification as described in Example 5.

UPLC-SEC was performed using a Waters BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 μm particles) set at 30 °C and mounted on a Waters Acquity UPLC system with a PDA detector. The run time consisted of 7 minutes and a total volume per injection of 2.8 mL with Hyclone DPBS/Modified-Calcium-Magnesium (Part No. SH30028.02) running buffer at 0.4 ml/min. Elution was monitored by UV absorbance in the range 200-400 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Empower 3 software.

6 shows UPLC-SEC results for a representative WT Fab heterodimer pair (containing a FLAG tag on the L chain) as well as a representative (H1L1 Fab component of LCCA designs 9735, 9737, and 9740) for designed Fab heterodimer pairs. show the profile. In general, the designed Fab heterodimer pairs showed similar UPLC-SEC profiles compared to WT.

Example 9: Evaluation of preferential pairing of heterodimers in co-expression sets comprising constant domains or constant and variable domain modifications in a bispecific antibody format

Heterodimeric designs were evaluated to determine if they also allow preferential pairing in a bispecific antibody format. In this example, to facilitate heterodimerization of native heavy chains, the Fc region of the full-length heavy chain of each heterodimer is asymmetrically modified so that one heavy chain contains the mutations T350V, L351Y, F405A and Y407V and the other heavy chain contained the mutations T350V, T366L, K392L and T394W (EU numbering).

Preparation of Constructs:

The heterodimer design was 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 composed of human IgG1 and mouse Fv regions. Constructs encoding D3H44, trastuzumab and cetuximab IgG heavy and light chains, including amino acid modifications according to design, were prepared as follows. Base DNA sequences for the heavy and light chains of D3H44, Trastuzumab and Cetuximab are shown in Table 3C. The D3H44, trastuzumab and cetuximab light chain sequences were prepared as described in Example 3, except that some sequences lacked the tag whereas others contained FLAG or HA tags. The D3H44, trastuzumab and cetuximab heavy chain sequences were prepared as described in Example 3, except that the full-length heavy chain was created by appending an IgG1*01 DNA sequence encoding the hinge-CH2-CH3 domain and the CH1 of the Fab heavy chain. Modifications were made to promote heterodimerization on the C-terminus of the domain. During annotation, canonical C-terminal heavy chain lysine residues were 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 the heterodimeric co-expression set design to pair preferentially to form a bispecific antibody was evaluated as described below. The assay is based on co-expression of the four chains (H1 and L1 chains from one antibody and H2 and L2 chains from another antibody) and detection of the presence of correctly formed bispecific antibodies using mass spectrometry (LC-MS). 8 provides a schematic depicting the potential products resulting from the co-expression of four initiating polypeptide chains and the absence of preferential pairing between the heavy and light chains of a heterodimer pair. The two full-length heavy chain constructs were co-expressed with the two native light chain constructs, resulting in 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 correctly paired bispecific antibodies (see Figure 8). Relative pairing specificity with respect to the amount of the preferred species H1-L1:H2-L2 versus others was determined using LC-MS after pA purification and deglycosylation. Where possible, chains were left untagged and all Mab and half-Ab species provided differed from each other by at least 50 Da. If mass differences prevented this possibility, an N-terminal tag (HA or FLAG) was added to the light chain to provide sufficient mass differentiation between species.

This assay involved in the expression and screening steps of the bispecific antibody is referred to as SMCA.

Mass spectrometry method

The degree 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. As the D3H44/trastuzumab heterodimer contains only Fc N-linked glycans, this system was treated with only one enzyme, N-glycosidase F (PNGase-F). Purified samples were de-glycosylated with PNGaseF as follows: 0.2 U PNGaseF/μg of antibody in 50 mM Tris-HCl pH 7.0, incubated overnight at 37°C, final protein concentration of 0.5 mg/mL. For the D3H44/Cetuximab and Trastuzumab/Cetuximab systems, due to the additional N-linked glycan in the Fab region of Cetuximab, the system is treated with N-glycosidase F plus numerous exoglycosidases It became. Typically, a mixture of 4 enzymes was 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 to a uniform core structure, M3F (GlcNAc ₂ Man ₃ Fuc ₁ ). Purified samples were de-glycosylated with a 4 enzyme mixture as follows: 0.2 U PNGaseF/μg antibody, 0.002 U α-neuraminidase/μg antibody, 0.0001 U β-galacto in 50 mM Tris-HCl pH 7.0. Sidase/μg of antibody and 0.2U β-N-acetylglucosaminidase/μg of antibody, incubated overnight at 37°C, final protein concentration of 0.5 mg/mL. However, in some cases, a three-enzyme treatment (N-glycosidase F, β-galactosidase and neuraminidase) was preferred to avoid mass overlap of sample components in LC-MS analysis. In these cases the Fab glycan was reduced to a slightly larger structure GOF (Man ₃ GlcNAc ₂ Fuc ₁ GlcNAc ₂ ). Purified samples were de-glycosylated with the 3 enzyme mixture using the same concentrations and conditions as described for the 4 enzyme mixture. After deglycosylation, samples were stored at 4 °C prior to LC-MS analysis.

Deglycosylated protein samples were analyzed by intact 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). Samples (5 μg) were injected onto a 2.1 x 30 mm Poros R2 reverse phase column (Applied Biosystems) and resolved using the following gradient conditions: 0-3 min: 20% Solvent B; 3-6 min: 20-90% solvent B; 6-7 min: 90-20% solvent B; 7-9 min: 20% solvent B. Solvent A was degassed with 0.1% formic acid aq. and solvent B was degassed with acetonitrile. The flow rate was 3 mL/min. The flow rate was split post-column to direct 100 μL into the electrospray interface. The column was heated to 82.5 °C and the solvent was heated full-column to 80 °C to improve the protein peak shape. The LTQ-Orbitrap XL was calibrated using ThermoFisher Scientific's LTQ cationic ESI calibration solution (caffeine, MRFA and Ultramark 1621) and calibrated using a 10 mg/mL solution of CsI. The cone voltage (source fragmentation setting) was 40 V, the FT resolution was 7,500 and the scan range was m/z 400-4,000. The LTQ-Orbitrap XL was tuned for optimal detection of larger proteins (>50 kDa).

Ranges containing multiple charged ions from full-size antibodies (m/z 2000-3800) and half-antibodies (m/z 1400-2000) can be obtained using MassLynx's MaxEnt 1 module, instrument control and data analysis software (Waters) It was separately deconvoluted with molecular weight profiles using . Briefly, the crude protein LC-MS data was first opened in QualBrower, Xcalibur's spectral viewing module (Thermo Scientific) and converted to MassLynx compatibility using the file conversion program provided by Databridge, Waters. Transformed protein spectra were viewed in MassLynx's Spectrum module and deconvolved using MaxEnt 1. The abundance of the different antibody species in each sample was determined directly from the obtained molecular weight profiles.

Representative design for SMCA assay

A total of 25 representative designs with high average LCCA performance values were selected from test clusters 1 to 12 in the SMCA format. Representative designs were selected based on a set of corresponding designs that share similar mutations while occupying similar spaces, using similar drivers. At least one representative design was selected from each cluster. Some clusters were represented in one representative design (i.e. clusters 1, 5, 7, 8, 10). The remaining clusters had more than 1 representative design, as clusters consisted of large (ie cluster 2) or small clusters (ie clusters 3, 4, 6, 9, 11 and 12). Although the designs within each cluster share sequence similarity, within a cluster the small clusters differ in at least one set of driver mutations. For clusters that were made up of small clusters, additional representative designs were selected from each small cluster.

Amino acid substitutions for each cluster are listed in Tables 15-27 and the corresponding representation for each cluster/small cluster is indicated. For cluster 1, only one design (9134-9521) was chosen to represent clusters occupying similar spaces as these designs use similar electrostatic actuators (see Table 15). For all members of this cluster, H1 was designed to allow negatively charged substitutions (L124E and Q179E) to form salt bridges with L1 positively charged substitutions (S176R and S131K or S131R). H2 was designed to allow positively charged substitutions (L124R and Q179K or S186K) to form salt bridges with L2 negatively charged substitutions (S176D and T178D or T178E and/or T180E). Mismatched pairing of H1L2 and H2L1 will be disfavored primarily due to electrostatic repulsion.

For cluster 2, two representative designs (9279-9518 and 9286-9402) were selected to represent large clusters (see Table 16). Within these clusters, the design used similar electrostatic actuators occupying similar space. For all members of this cluster, H1 is designed to accept negatively charged substitutions (L124E and L143E or L143D), resulting in L1 positively charged substitutions (S176R and (Q124K and/or T178K) or (Q124K and Q160K) combination) and formed a salt bridge. H2 is designed to allow positively charged substitutions (L124R and Q179K or S186K or S186R) to form salt bridges with L2 negatively charged substitutions (S176D and T178D or T178E and/or T180E). Mismatched pairing of H1L2 and H2L1 will be disfavored primarily due to electrostatic repulsion.

For cluster 3, 5 representative designs (9338-9748, 9815-9825, 6054-9327, 9066-9335 and 9121-9373) were selected to represent each of the 5 small clusters (see Table 17). All members of this cluster utilized similar electrostatic drivers for H1 (L124E), L1 (S176R), H2 (L124R), and L2 (S176D), preferentially allowing the formation of salt bridges in paired heterodimers. whereas mismatched pairs will be disfavored mainly due to electrostatic repulsion. Design 6054-9327 was chosen to represent this small cluster, to represent a design using primarily constant region drivers. In addition to these electrostatic interactions, one small cluster also contained variable region steric drivers (H1 L45P and L1 P44F) and therefore representatives containing these variable region drivers were selected to represent this small cluster (9338-9748) . Another small cluster also contained variable region electrostatic drivers in all Fab heterodimers (H1 Q39E, L1 Q38R, H2 Q39R, L2 Q38E) and therefore a representative containing these variable region drivers was selected and this small cluster (9815- 9825). Moreover, one small cluster consists of one member and therefore one representative design (9066-9335) contains a disulfide engineered between H1 F122C and L1 Q124C. The remaining small clusters, denoted 9121–9373, use the constant region driver primarily with the additional substitutions H1 to H172T and L1 to S174R to slightly alter the interaction of the H1L1 constant region driver, while also probing the effect of H172R in HC2. did

For cluster 4, two representative designs (9168-9342 and 9118-6098) were selected to represent each of the two small clusters (see Table 18). All members of this cluster utilized similar electrostatic drivers on H1 (L124E), L1 (S176R or S176K), H2 (L124R), and L2 (S176D), preferentially inhibiting the formation of salt bridges in paired heterodimers. While permissive, mismatched pairs will be disfavored primarily due to electrostatic repulsion. One small cluster, denoted 9118-6098, primarily utilizes the shared electrostatic driver for preferential pairing, while another small cluster, denoted 9168-9342, allows the formation of additional salt bridges H1 (K228D) and L1 (S121K). ) was further used.

Cluster 5, denoted by unique identifiers 9116-9349, consists of only one member (see Table 19). This design includes all electrostatic actuators on H1 (L124E), L1 (S176R), H2 (L124R) and L2 (S176D) as well as solid actuators on H1 (A139W), L1 (F116A_V133G_L135V), H2 (A139G_V190A) and L2 (V133G_L135W). was used. As a result, preferentially for pairwise heterodimers, charged substitutions will allow the formation of salt bridges. Regarding mispaired Fab heterodimers, formation will be disfavored due to electrostatic repulsion as well as additional steric effects.

For cluster 6, two representative designs were selected to represent each of the two small clusters (see Table 20). All members of this cluster have similar electrostatic drivers in the constant regions (Q179E on H1, S131K on L1, S186R on H2, and Q124E, Q160E, and T180E on L2) that preferentially allow the formation of salt bridges in pairwise heterodimers. while mismatched pairs will be disfavored mainly due to electrostatic repulsion. In addition, small clusters were also composed of different variable region drivers. One small cluster, denoted by unique identifiers 9814-9828, utilized electrostatic drivers in the variable region (Q39E on H1, Q38R on L1, Q39R on H2, and Q38E on L2). Another small cluster utilized variable domain stereo actuators consisting of L45P in H1 and P44F in L1. As a result, for such small clusters, mismatched pairs will be further disfavored due to the steric effect introduced. Note that this small cluster represents a design derived from unique identifiers 9745-9075, which differs only in the absence of Q38E on L2.

For cluster 7, only one design (9060-9756) was chosen to represent the cluster as these designs use similar electrostatic and stereo actuators (see Table 21). The shared electrostatic drivers included L143E and Q179E on H1, Q124R on L1, Q179K on H2, and Q124E, Q160E, and T180E on L2. The shared stereo actuators included A139W on H1, F116A_L135V on L1, and L135W on L2. As a result, preferentially for pairwise heterodimers, charged substitutions in the Fab region will allow the formation of salt bridges. Regarding mispaired heterodimers, formation will be disfavored due to electrostatic repulsion as well as additional steric effects.

For cluster 8, only one design (9820-9823) was chosen to represent the cluster as these designs used similar electrostatic actuators (see Table 22). In the variable region, Q39E on H1, Q38R on L1, Q39R on H2, and Q38E on L2 were used. In the constant region, L143E on H1, Q124R, Q160K and T178R on L1, Q179K on H2, and Q124E, Q160E and T180E on L2 were used. For preferentially paired heterodimers, charged substitutions in the Fab region allow the formation of salt bridges whereas for mispaired heterodimers, formation will be disfavored primarily due to electrostatic repulsion.

For cluster 9, two representative designs were selected to represent each of the two small clusters (see Table 23). All members of this cluster have similar electrostatic drivers in the constant regions (L143E on H1, Q124R on L1, Q179K on H2, and Q124E, Q160E, and T180E on L2) that preferentially allow the formation of salt bridges in pairwise heterodimers. while mismatched pairs will be disfavored mainly due to electrostatic repulsion. Also, the small cluster differs in the presence/absence of variable region drivers (L45P on H1 and P44F on L1). As a result, for small clusters containing variable region drivers, mismatched pairs will be further disfavored due to the steric effect introduced. A representative design for this small cluster containing variable region drivers was derived from unique identifiers 9751-9065, which differed only in the absence of Q38E on L2. For small clusters lacking variable region substitutions, a representative design is 9611-9077.

For cluster 10, only one design (9561-9095) was chosen to represent the cluster as these designs use similar electrostatic and stereo actuators occupying similar space (see Table 24). Shared electrostatic drivers included L143E and Q179E on H1, similarly positioned Q124R, Q124K or S131K on L1, Q179K on H2, and Q124E and T180E on L2. The shared stereo actuators included L124W on H1, V133A on L1, and V133W on L2. As a result, preferentially for pairwise heterodimers, charged substitutions in the Fab region will allow the formation of salt bridges. Regarding mispaired heterodimers, formation will be disfavored due to electrostatic repulsion as well as additional steric effects.

For cluster 11, three designs (9049-9759, 9682-9740 and 9667-9830) were selected to represent each of the three small clusters (see Table 25). All members of this cluster drive preferential pairing of heterodimers using electrostatic displacement. As a result, preferentially for pairwise heterodimers, charged substitutions in the Fab region will allow the formation of salt bridges. Regarding mispaired heterodimers, formation will be disfavored primarily due to electrostatic repulsion. For the small cluster represented by unique identifiers 9667-9830, the shared substitutions are negatively charged substitutions on H1 (L143E or L143D and Q179E or Q179D), (T178R or T178K) positively charged substitutions on L1, positively on H2. A charged substitution (S186K or S186R or Q179K or Q179R) and a negatively charged substitution on L2 (Q124E) were included. Another small cluster, represented as the only member of this small cluster (unique identifiers 9049-9759), additionally contained substitutions for the formation of engineered disulfide bonds. The remaining clusters, denoted by unique identifiers 9682-9740, used similar drivers for H1 and L1 as the other two smaller clusters; However, different constant region H2 and L2 drivers were used. H2 utilized L143R or L143K and L2, in addition to the Q124E substitution (shared with two other small clusters), V133E or V133D.

For cluster 12, 4 designs (9696-9848, 9986-9978, 9692-9846 and 9587-9735) were selected to represent each of the 4 small clusters (see Table 26). All members of this cluster drive preferential pairing of heterodimers using electrostatic displacement. Some members additionally utilized steric actuators. Small clusters denoted by unique identifiers 9696-9848 utilized all electrostatic and steric actuators. The shared electrostatic substitutions within this small cluster consisted of L143E on H1, Q124R and T178R on L1, similarly located S186K or S186R or Q179K or Q179R on H2, and Q124E and T180E on L2. The shared steric substitutions within this small cluster consisted of S188L on H1 and S176L or V133Y or V133W on L2; For designs using V133Y or V133W on L2, L143A or L124A were also present on H2 to accommodate the bulk mutation. For the small clusters represented by unique identifiers 9692-9846, similar electrostatic actuators were used for comparison with the small clusters represented by unique identifiers 9696-9848; For some members, a similarly positioned substitution, T178E, was used instead of T180E on L2. Moreover, a subset from this small cluster also utilized similar steric drivers with similarly positioned substitutions of T178Y or T178F for S176L on L2. The small clusters indicated by unique identifiers 9986-9978 only drove preferential pairing using electrostatic actuators. Similar shared substitutions were used for H1, L1 and H2; However, a different L2 substitution (S131E) was used. The remaining small clusters, denoted by unique identifiers 9587-9735, used similar electrostatic actuators on H1 and L1 (but on L1 T178R was not used on all members within this small cluster); However, different electrostatic actuators were used for H2 (L143R or L143K) and L2 (Q124E and V133E or Q124E and V133D). A couple of members within this small cluster also utilized a similar steric actuator consisting of S188L on H1 and S176L on L2. Overall, preferentially for pairwise heterodimers, charged substitutions in the Fab region will allow the formation of salt bridges. Regarding mispaired heterodimers, formation will be disfavored due to electrostatic repulsion. Moreover, for designs that also use stereoscopic actuators, the formation will be further disfavored due to stereoscopic effects. Cluster 13 consists of one member, 9122-9371 (see Table 27). This design utilized a disulfide engineered between H1 F122C and L1 Q124C as a covalent driver for preferential pairing of heterodimers. In addition, since the design also lacks native interchain disulfide, the formation of disulfide bonds was confirmed with non-reducing and reducing SDS-PAGE gels. These designs have not been tested in the SMCA format; However, engineered disulfides were tested in the presence of natural interchain disulfides and in combination with additional constant region drivers (Cluster 3, representative design 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 at a density of 1.7 - 2 x 10 ⁶ cells/ml were grown in FreeStyle(TM) F17 medium (Invitrogen cat# A) supplemented with 4 mM glutamine and 0.1% Pluron F-68 (Invitrogen cat# 24040-032). -1383501) at 37°C. A total volume of 50 ml was transfected with a total of 50 ug DNA using PEI-Pro (Polyplus cat# 115-010) at a DNA:PEI ratio of 1:2.5. 24 hours after addition of the DNA-PEI mixture, cells were transferred to 32° C. and incubated for 7 days prior to harvest. Culture medium was harvested by centrifugation and vacuum filtered using a Steriflip 0.2 μM filter. The filtered culture medium was then purified using Protein A MabSelect SuRe resin (GE Healthcare #17-5438-02) as follows. The filtered culture medium was applied to a column (Hyclone DPBS/modified, calcium free, magnesium free, # SH-300028.02) that had previously been equilibrated with PBS. Heterodimeric antibody species were then washed with PBS and eluted with 100 mM citrate pH 3.6 on an Amicon Ultra 15 centrifuge filter Ultracell 10K (Millipore # SCGP00525). Buffer was then exchanged to PBS and samples were calibrated prior to deglycosylation and LC-MS.

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

For the testing of each of the 25 representative designs within each bispecific Ab system, the H1:H2:L1:L2 DNA ratios used yielded the majority of the bispecific Ab species while yielding low amounts of the half Abs. from the corresponding wild-type bispecific system with (see Tables 32a, b and c). For the D3H44/trastuzumab system, the ratios used were 15 (H1), 15 (H2), 53 (L1), 17 (L1), where H1 and L1 refer to D3H44 and H2 and L2 refer to tra mention stuzumab. For the Trastuzumab/Cetuximab system, the ratios used were 15 (H1), 15 (H2), 17 (L1), 53 (L2), where H1 and L1 refer to Trastuzumab and H2 and L2 refers to cetuximab. For the D3H44/cetuximab system, the ratios used were 15 (H1), 15 (H2), 53 (L1), 17 (L2), where H1 and L1 refer to D3H44 and H2 and L2 refer to cetux. mentions simab.

Moreover, the design was tested in all orientations for the D3H44/cetuximab and trastuzumab/cetuximab bispecific systems, such that in one orientation, the substitutions present on H1L1 and H2L2 are the same for the bispecific Ab system, respectively. were tested on antibody 1 (Ab1) and antibody 2 (Ab2) of , and in another "inverted" orientation, the substitutions present on H1L1 and H2L2 were tested on Ab2 and Ab1, respectively (see Tables 28 a and b). The "_1" appended to the unique identifier indicates a design in which the heavy chain and related light chain substitutions were placed on the antibody, which gave a stronger LCCA preferential pairing result (see Table 13a), wherein the heavy chain is its value compared to the light chain from another antibody. There was weak competition for the associated light chain. The "_2" appended to the unique identifier indicates the opposite "flipped" orientation in which the heavy chain and related light chain substitutions are located on the antibody, giving the stronger LCCA preferential pairing results (see Table 13a), wherein the heavy chain is separated from the other antibody. It competed more strongly for its related light chain compared to the light chain. For the D3H44/Trastuzumab system, the design was only tested in the "_1" orientation (see Table 28c).

SMCA results

The D3H44/trastuzumab system was treated with only one enzyme (PNGase-F) and was completely deglycosylated. For multi-enzyme treatment, the attached saccharide in the Fab region was usually cleaved into core M3F (using 4 enzyme treatment) or GOF (using 3 enzyme treatment). Overall, in most cases, the deglycosylation treatment resulted in the ability to identify all possible different species identified by LC-MS. In many cases, each species is represented by a single LC-MS peak. Exceptions include side peaks that also correspond similarly to the desired bispecific species (possibly heterologous in cleavage of the adduct or leader peptide); However, due to the ambiguity of the side peaks, these side peaks were not considered in the contribution to the bispecific species. In addition, some designs within the D3H44/cetuximab (3519_1, 3522_1) and trastuzumab/cetuximab (9748-9338_1) systems required multiple peaks to account for the species due to the variability of the high mannose attached. All of these designs introduced glycosylation sites in the cetuximab light chain. Note that in some cases it was not possible to discriminate between some small species (comprising less than 5% of all species) due to low mass separation (i.e. less than 50 Da difference) between species. Moreover, the desired bispecific species, H1-L1_H2-L2, cannot usually be experimentally identified on the basis of LC/MS from the mispaired type: H1-L2_H2-L1. As such, when bispecific content is reported in the table, the absence of mispaired species of this type cannot be entirely excluded. However, the very low content observed for species such as H1-L2_H1-L2 and H2-L1_H2-L1 as well as the H1-L2 and H2-L1 half antibodies indicates only minor if any contamination of the bispecific species occurs.

LC-MS data are shown in Tables 29a, 29b and 29c. For comparison, wild-type data are also shown in Tables 33a, 33b and 33c and are marked with "NA" in the "Cluster" column as well as the "SMCA Unique Identifier" column. All three bispecific wild-type systems exhibited a lopsided bias whereby one light chain led to binding to all heavy chains (see Table 33 and Figure 9). Moreover, at least in the trastuzumab/cetuximab system, tag placement also appears to have a significant effect on H1L1 and H2L2 pairing. Therefore, to evaluate the effect of the design on the percentage and mobility of the desired bispecific species versus wildtype, a comparison to the corresponding wildtype bispecific construct at the same H1:H2:L1:L2 DNA ratio is performed below. and reported: “Change in % H1L1 pairing (across all H1 species) relative to wild type”, “Change in % H2L2 pairing (across all H2 species) relative to wild type” and “H1:H2 relative to wild type: % change in L1:L2" (considering full-scale antibody species alone) column (see Table 29). Note that for evaluation of % H1L1 pairing (across all H1 species) or % H2L2 pairing (across all H2 species), all species are evaluated for pairing in the Fab region. If the corresponding wild-type bispecific construct was not evaluated by SMCA, a similar wild-type construct was compared. Estimates are indicated with "***" next to the reported value. Similar wild-type constructs selected for comparison were selected as follows. To assess mobility, each wild-type construct was presented in a SMCA experiment (performed at different ratios) showing the highest "% H1L1 and % H2L2 pairing (across all species)". In order to evaluate the effect of the design on the percentage of the desired bispecific species versus wildtype, each wildtype construct (performed at different ratios) exhibited the highest % of H1:H2:L1:L2 (considering the full-size antibody species alone). ) as shown in the SMCA experiment. In all cases, the median value, in addition to all wild-type constructs within the bispecific system, was then selected as the wild-type value for comparison.

For each design, mobility was assessed by increasing attention in total H:L pairing across all species relative to WT, specifically in % H1L1/all H1 species and/or % H2L2/all H2 species. In addition, the effect on the percentage of the target bispecific species was also evaluated, focusing only on the full-size antibody species for comparison, as half antibodies, if present, by preparative SEC or additional H1:H2:L1:L2 It can be eliminated/minimized through DNA titration. Table 30 a, b and c show that preparative SEC can be effective in removing/minimizing half Ab species. Table 32 a, b and c show that the percentage of half Ab species can also be processed during transfection using various DNA titration ratios.

For the D3H44/cetuximab system (Table 29a), all but one design (9327-6054_2) migrated across all species relative to the wild type as assessed by H1L1 pairing. The majority of designs (except 9327-6054_2 and 9134-9521_2) also showed an increased percentage of the bispecific antibody of interest when only total Ab species were considered. Moreover, designs reduced the observed primary mispaired antibody species (H1H2L2L2) relative to the wild type, except for one design that did not migrate (9327-6054_2). In addition, designs migrated to all orientations, except for 9327-6054_2 and the corresponding design 9327-6054_1 in other orientations, and the majority of designs showed similar effective H:L pairing in all orientations.

For the D3H44/Trastuzumab system (Table 29b), all designs migrated across all species relative to wildtype when evaluated by H1L1 pairing. In addition, all designs showed an increased percentage of the bispecific antibody of interest (when considering the total Ab species alone). Moreover, most designs reduced the primary mispaired antibody species (H1H2L2L2) observed relative to the wild type. However, note that no data were reported for 9611-9077_1 (Table 28c) due to lack of expression.

Regarding the trastuzumab/cetuximab system (Table 29c), at least 35 out of 49 designs showed mobility when assessed by H2L2 pairing across all H2 species ("for wildtype (across all H2 species) ) % change in H2L2 pairing” positive values in the column). Designs that did not appear to migrate include: 9279-9518_2, 3522_2, 9815-9825_2, 9327-6054_2, 9118-6098_2, 9748-9338_2, 9692-9846_2, 9587-9735_2, 9814-9829_92, 3514_2, 3519_96 9978_2, 9168-9342_2 and 9066-9335_1 (negative values in column "change in % H2L2 pairing (across all H2 species) relative to wild type"); However, the design with other orientations did not show portability (note that 9279-9518_1 was not tested due to lack of samples). All designs that showed mobility showed a reduced percentage of the primary mispaired antibody species (H1H2L1L1) observed in wild-type experiments. Also, of the designs that were moved, only 2 designs (9134-9521_1 and 9279-9518_2) showed a reduced percentage of the bispecific Ab of interest when only total antibody species were considered compared to wild type.

In general, most designs that increased the H:L pairing of the weaker competing antibody resulted in an increased percentage of the desired bispecific antibody (consider full-size antibody alone). Regarding the orientation, most designs in the "_1" orientation exhibited similar or better mobility compared to H:L pairing compared to the "_2" orientation (mainly observed in the trastuzumab/cetuximab system). exception). Moreover, Table 35a lists designs that migrated in all orientations across all 3 tested bispecific systems (D3H44/Cetuximab, D3H44/Trastuzumab, and Trastuzumab/Cetuximab). Table 35b shows migration in one orientation across all three bispecific systems (D3H44/Cetuximab, D3H44/Trastuzumab, and Trastuzumab/Cetuximab) and the other orientation for only one bispecific system. List the designs moved to . Also, with the orientation specified, the same mutations are present on the heavy chain and the weaker competing mobilization light chain in all three bispecific systems, and the light chain utilization is at least 10% greater.

Regarding the mobility and performance of the clusters, for the D3H44/trastuzumab bispecific system, all members in all clusters showed mobility (see Fig. 11a) and the bispecific antibody of interest (considering the full-size antibody alone) increased the percentage of (see Fig. 11b). For the D3H44/cetuximab bispecific system, all clusters showed mobility and only one member in cluster 3 that showed reduced H1L1 pairing across all H1 species compared to wild type (see Fig. 11c). . In addition, all clusters included members that showed an increase in the percentage of the bispecific antibody of interest relative to the wild-type (considering the full-size antibody alone); However, cluster 3 (clusters 1, 3 and 4) also contains members that showed a decrease in the percentage of the bispecific antibody of interest relative to the wild type (consider full-size antibody alone) (see Figure 11d). Regarding the trastuzumab/cetuximab bispecific system, all clusters contain variants exhibiting design mobility; However, only a few clusters (1, 5, 7, 8, 10, 11) contain variants in which all individual members exhibit mobility (see Fig. 11e). In addition, all clusters contained constructs showing an increased percentage of the bispecific antibody of interest relative to the wild-type (consider full-size antibody alone) (see Fig. 11f). For clusters in which all members showed mobility, all members in clusters 5, 7, 8, 10 and 11 also showed an increase in the percentage of the desired bispecific antibody relative to the wild type (consider full size antibody alone).

Overall, when all 3 bispecific systems are considered together, all members within clusters 1, 5, 7, 8, 10, and 11 showed mobility (see Figure 11g); Clusters 5, 7, 8, 10, and 11 all showed an increase in the percentage of the bispecific antibody of interest relative to the wild type (considering the full-size antibody alone) (see Figure 11h).

Overall, most designs that increased the H:L pairing of the weaker competing antibody resulted in an increased percentage of the desired bispecific antibody (considering the full-size antibody alone). Regarding orientation, most designs with "_1" orientation showed similar or better mobility compared to H:L pairing compared to "_2" orientation (mainly observed in trastuzumab/cetuximab systems). exception).

Example 10: Parental Mabs for Preparative Size Exclusion Chromatography (SEC) and Biophysical Characterization of Selected SMCA Bispecific Heterodimeric Antibodies.

정화기 시스템상에 실장된 Superdex 200 10/300 GL (GE Healthcare) 칼럼을 이용하여 분리되었다. PBS (Hyclone DPBS/변형된, 무 칼슘, 무 마그네슘, Cat no SH-300028.02)내 이종이량체 항체 샘플 (0.3-0.5 ml)은 PBS로 충전된 0.5ml 루프에 수작업으로 로딩되었다. 샘플은 그 다음 칼럼상에 자동으로 주사되었고 1 CV 용출 용적으로 0.5ml/min에서 분해되었다. 단백질 용출은 OD₂₈₀에서 모니터링되었고 0.5 ml 분획에 수집되었다. 각 SMCA 샘플에 대하여, 주요 피크를 포함하는 분획은 풀링되었고 추가로 생체물리학적으로 특성화되었다.A subset of SMCA samples were selected for further biophysical characterization. Most of these SMCA samples typically showed high pairing (>80% pairing in the H1L1+H2L2/all species column) and low amounts of half antibody species (<30% considering all half antibody species) . Preparative SEC was performed as follows. Heterodimeric antibody samples were from Pharmacia (GE Healthcare)

Separation was performed using a Superdex 200 10/300 GL (GE Healthcare) column mounted on a purifier system. Heterodimeric antibody samples (0.3-0.5 ml) in PBS (Hyclone DPBS/modified, calcium free, magnesium free, Cat no SH-300028.02) were manually loaded into a 0.5 ml loop filled with PBS. Samples were then automatically injected onto the column and resolved at 0.5 ml/min with 1 CV elution volume. Protein elution was monitored at OD ₂₈₀ and collected in 0.5 ml fractions. For each SMCA sample, the fraction containing the main peak was 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 analyzed for preferential pairing of bispecific heterodimeric antibodies using the LC-MS method as described in Example 9. All these samples show an enrichment in the percentage of the desired bispecific antibody species as well as a decrease in the percentage of half antibody species (Tables 29 and 30).

Example 12: Thermal stability of SMCA bispecific heterodimeric antibodies.

After preparative SEC, the thermal stability of the selected SMCA bispecific heterodimeric antibodies was measured and compared to that of the parental D3H44 and trastuzumab monoclonal antibodies as well as the cetuximab 1 armed antibody. In general, a one-armed antibody has one full-length heavy chain, one truncated heavy chain lacking the Fab region (and incorporating the C233S substitution) and one heavy chain heterodimerization achieved as described in Example 9. It refers to constructs consisting of light chains.

Measurement of thermal stability

The thermal stability of selected bispecific heterodimeric antibodies and wild-type controls was determined using differential scanning calorimetry (DSC) as follows. After preparative SEC treatment, 400 μL samples, primarily at concentrations of 0.2 mg/ml or 0.4 mg/mL in PBS, were subjected to DSC analysis with a VP-capillary DSC (GE Healthcare). At the start of each DSC run, 5 buffer blank injections were performed to stabilize the baseline, and buffer injections were placed prior to each sample injection for reference. Each sample was scanned from 20 to 100 °C at a rate of 60 °C/hr with low feedback, 8 sec filter, 5 min preTstat, and 70 psi nitrogen pressure. The obtained thermograms were referenced and analyzed using Origin 7 software.

Results are shown in Tables 31a, b and c. Fab Tm values reported in the table for wild type were obtained for the homodimeric antibodies for D3H44 (79 °C) and Trastuzumab (81 °C) and for the one-armed antibody for cetuximab (72 °C). It became. For the WT D3H44/cetuximab and trastuzumab/cetuximab heterodimeric antibodies, only 2 peaks corresponding to the Fab Tms are observed. No different peaks are observed for CH2 (due to overlap with cetuximab Fab) or CH3 (due to overlap with Tm values of D3H44 and Trastuzumab Fabs). For the WT D3H44/Trastuzumab heterodimeric antibody, as the Tm values of the two Fabs from D3H44 and Trastuzumab are similar, the peak at approximately 72°C corresponds similarly to all Fabs at 81°C. corresponds to CH2.

In Tables 31a, b and c, the Tm value(s) of only the peak(s) corresponding to all Fabs are reported unless otherwise specified. Also note that for some heterodimer samples, the protein concentration was low (less than 0.4 mg/mL) leading to increased noise at baseline. As a result, in the D3H44/trastuzumab system, some samples obtained DSC curves with low peak intensities, making it difficult to discriminate between the CH2 peak and possibly the destabilized Fab. In these cases, Tm values between 70 and 72 °C are also reported (Table 31a). Overall, most heterodimeric antibodies exhibit similar thermal stability (up to 3 °C) to the corresponding wild-type molecule. Moreover, most heterodimeric antibodies show no additional peaks to suggest significant destabilization of the CH2 or CH3 peaks. One exception includes heterodimeric antibody 9611-9077_2 engineered from the trastuzumab/cetuximab system which exhibits an additional peak at 60° C., which can be attributed to CH2 destabilization.

Example 13: Determination of Antigen Affinity of Bispecific Heterodimeric Antibodies

The ability of the bispecific antibodies to bind related antigens was evaluated to determine whether amino acid substitutions had any effect on antigen binding. 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 1 M 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, CA). PBS running buffer with 1% Tween20 (PBST) is from Teknova Inc. (Hollister, CA). Goat polyclonal anti-human Fc antibodies were prepared by 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 plasmon resonance instrument (GE Healthcare Life Science, Mississauga, ON) with PBST running buffer (with 0.5 M EDTA stock solution added to a final concentration of 3.4 mM) at a temperature of 25°C. was performed by An anti-human Fc capture surface was created using a Series S sensor chip CM5 using default parameters under the immobilization wizard in Biacore T200 control software set to a target of 2000 resonance units (RUs). Screening of antibody variants for binding to the Her2 ECD, TF or EGFR ECD antigen target occurred in two steps: anti-human Fc antibody indirect capture of antibody variants on the flow cell surface followed by kinetic analysis using a single cycle kinetics methodology. Injection of 5 concentrations of purified antigen. Capture variants or controls were injected at 1 μg/mL across individual flow cells for 60 s at a flow rate of 10 μL/min. In general, this resulted in entrapment of approximately 50-100 RUs on the anti-human Fc surface. The first flow cell was emptied to use as a blank control. This capture step was followed immediately by a dissociation phase of 300 s for EGFR ECD, 1800 s for Her2 ECD, and 3600 s for TF, followed by 180 s of antigen sequentially injected across all 4 flow cells at 100 μL/min. Five concentrations (5 nM, 2.5 nM, 1.25 nM, 0.63 nM and 0.31 nM for TF or EGFR ECD antigens, or 40 nm, 20 nm, 10 nm, 5 nm, and 2.5 nm for Her2 ECD antigens) were followed. The captured antibody surface was regenerated with 10 mM glycine pH 1.5 for 120 s at 30 μL/min to prepare for the next injection cycle. At least 2 mock-buffer injections were performed for each analyte injection used for reference. The obtained single cycle kinetic sensorgrams were analyzed using Biacore T200 BiaEvaluation software and fitted to a 1:1 binding model.

The antigenic affinity of the heterodimeric antibody was evaluated with reference to the respective wild-type controls: Mab against D3H44, Trastuzumab OAA and Cetuximab OAA. Antigen affinities were also obtained for wild-type bispecific antibodies; However, SPR entrapment of WT bispecifics can be heterogeneous (eg including entrapment of mispaired heterodimers), thereby interfering with KD determinations (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 all D3H44/Trastuzumab and Trastuzumab/Cetuximab systems, antigen affinities were similar to the corresponding WT controls (see Tables 31a and c). Exceptions include 11 engineered antibodies that did not show Her2 binding. In all D3H44/Trastuzumab and Trastuzumab/Cetuximab systems, her2 binding was not observed for the six engineered heterodimeric antibodies, 9049-9759_1 and 9682-9740_1 and 3522_1. Moreover, for the trastuzumab/cetuximab system, five further engineered 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 11 engineered antibodies shared constant region mutations on the H chain (L143E_K145T) and L chain (Q124R_T178R). The other engineered antibody 9286-9402_2 shared the same constant region mutation on the H chain (L143E_K145T) and similar mutations on the L chain (Q124K and S176R).

Example 14: Ultra-Performance Liquid Chromatography Size Exclusion Chromatography (UPLC-SEC) Profiles of Engineered Heterodimeric Antibodies as well as Wild-Type Heterodimeric and Homodimeric Antibodies

After preparative SEC of the engineered heterodimeric antibodies as well as the control wild-type bispecific and homodimeric antibodies, UPLC-SEC was performed using a Waters BEH200 SEC column ( 2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 μm particles). The run time was 0.4 ml/min total per injection of 2.8 mL with working buffer of PBS and 0.02% polysorbate 20 or 20 mM NaPO4, 50 mM KCl, 0.02% polysorbate 20, 10% acetonitrile, pH 7. volume and 7 minutes. Elution was monitored by UV absorbance in the range 210-400 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Empower 3 software.

10 shows UPLC-SEC profiles to represent engineered heterodimeric antibodies as well as representative WT heterodimeric antibodies. In most cases, engineered heterodimeric antibodies have average percentages of monomers of 99.18%, 98.70% and 98.77% for D3H44/Trastuzumab, D3H44/Cetuximab, and Trastuzumab/Cetuximab, respectively. , which showed a UPLC-SEC profile similar to that of the corresponding WT heterodimeric antibody (see Tables 31a, 31b and 31c).

While the invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, it is understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention. It will be.

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

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Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 3 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 1 55 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 4 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Glu Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Asp Pro Glu Gln Gly Asn Thr Ile Tyr Asp Pro Lys Phe 50 55 60 Gln Asp Arg Ala Thr Ile Ser Ala Asp Asn Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Thr Ala Ala Tyr Phe Asp T yr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys P ro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys T hr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 5 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 S er Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 3 80 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <210> 6 <211> 448 <212> PRT <213> Artificial Sequence <220> < 223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Se r Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu T rp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 7 <211> 1347 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 7 gaggtgcagc tggtggaaag cggaggagga ctggtgcagc caggaggatc tctgcgactg 60 agttgcgccg cttcaggatt caacatcaag gacacctaca ttcactgggt gcgacaggct 120 ccaggaaaag gactggagtg ggtggctcga atctatccca ctaatggata cacccggtat 180 gccgactccg tgaaggggag gtttactatt agcgccgata catccaaaaa cactgcttac 240 ctgcagatga acagcctgcg agccgaagat accgctgtgt actattgcag tcgatgggga 300 gggacggat t ctacgctat ggattattgg ggacagggga ccctggtgac agtgagctcc 360 gcctctacca agggccccag tgtgtttccc ctggctcctt ctagtaaatc cacctctgga 420 gggacagccg ctctgggatg tctggtgaag gactatttcc ccgagcctgt gaccgtgagt 480 tggaactcag gcgccctgac aagcggagtg cacacttttc ctgctgtgct gcagtcaagc 540 gggctgtact ccctgtcctc tgtggtgaca gtgccaagtt caagcctggg cacacagact 600 tatatctgca acgtgaatca taagccctca aatacaaaag tggacaagaa agtggagccc 660 aagagctgtg ataagaccca cacctgccct ccctgtccag ctccagaact gctgggagga 720 cctagcgtgt tcctgtttcc ccctaagcca aaagacactc tgatgatttc caggactccc 780 gaggtgacct gcgtggtggt ggacgtgtct cacgaggacc ccgaagtgaa gttcaactgg 840 tacgtggatg gcgtggaagt gcataatgct aagacaaaac caagagagga acagtacaac 900 tccacttatc gcgtcgtgag cgtgctgacc gtgctgcacc aggactggct gaacgggaag 960 gagtataagt gcaaagtcag taataaggcc ctgcctgctc caatcgaaaa aaccatctct 1020 aaggccaaag gccagccaag ggagccccag gtgtacacac tgccacccag cagagacgaa 1080 ctgaccaaga accaggtgtc cctgacatgt ctggtgaaag gcttctatcc tagtgatatt 1140 gctgtggagt gggaatcaaa tggacag cca gagaacaatt acaagaccac acctccagtg 1200 ctggacagcg atggcagctt cttcctgtat tccaagctga cagtggataa atctcgatgg 1260 cagcagggga acgtgtttag ttgttcagtg atgcatgaag ccctgcacaa tcattacact 1320 cagaagagcc tgtccctgtc tcccggc 1347 <210> 8 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic polynucleotide <400> 8 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca ggacgttaac accgctgtag cttggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctattct gcatcctttt tgtacagtgg ggtcccatca 180 aggttcagtg gcagtcgatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag cattacacta ccccacccac tttcggccaa 300 gggaccaaag tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaagtg gataacgccc tccaatcggg taactcccaa 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag acga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642 <210> 9 <211> 1344 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 9 caggtgcagc tgaaacagag cggcccgggc ctggtgcagc cgagccagag cctgagcatt 60 acctgcaccg tgagcggctt tagcctgacc aactatggcg tgcattgggt gcgccagagc 120 ccgggcaaag gcctggaatg gctgggcgtg atttggagcg gcggcaacac cgattataac 180 accccgttta ccagccgcct gagcattaac aaagataaca gcaaaagcca ggtgtttttt 240 aaaatgaaca gcctgcagag caacgatacc gcgatttatt attgcgcgcg cgcgctgacc 300 tattatgatt atgaatttgc gtattggggc cagggcaccc tggtgaccgt gagcgcggcg 360 agcaccaaag gcccgagcgt gtttccgctg gcgccgagca gcaaaagcac cagcggcggc 420 accgcggcgc tgggctgcct ggtgaaagat tattttccgg aaccggtgac cgtgagctgg 480 aacagcggcg cgctgaccag cggcgtgcat acctttccgg cggtgctgca gagcagcggc 540 ctgtatagcc tgagcagcgt ggtgaccgtg ccgagcagca gcctgggcac ccagacctat 600 atttgcaacg tgaaccataa accgagcaac accaaagtgg ataaaaaagt ggaaccgaaa 660 agctgcgata aaacccatac ctgcccgccg tgcccggcgc cggaactgct gggcggcccg 720 agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa 780 gtgacctgcg tggtggtgga tgtgagccat gaagatccgg aagtgaaatt taactggtat 840 gtggatggcg tg gaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtataacagc 900 acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa 960 tataaatgca aagtgagcaa caaagcgctg ccggcgccga ttgaaaaaac cattagcaaa 1020 gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagccg cgatgaactg 1080 accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg 1140 gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg 1200 gatagcgatg gcagcttttt tctgtatagc aaactgaccg tggataaaag ccgctggcag 1260 cagggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag 1320 aaaagcctga gcctgagccc gggc 1344 <210> 10 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 10 gacatcctgc tgactcagag cccagtgatc ctgtcagtca gcccaggaga gcgggtgtcc 60 ttctcttgca gagcaagtca gtcaatcgga acaaatattc actggtacca gcagaggact 120 aacggctccc ctcgcctgct gattaagtat gctagcgaat ccatctctgg cattccatct 180 cggttcagtg gctcaggag cggaacagac tttactctgt ccatcaattc tgtggagagt 240 gaagacattg ccgattacta ttgccagcag aacaataact ggcccaccac attcggcgct 300 gggaccaagc tggagctgaa acgaacagtg gccgctcctt ctgtcttcat ctttccccct 360 agtgacgaac agctgaaaag cggcacagcc tccgtggtct gtctgctgaa taacttttac 420 ccaagagagg caaaggtgca gtggaaagtc gataatgccc tgcagtcagg gaacagccag 480 gagtccgtga ctgaacagga ctctaaggat agtacctatt cactgagctc cactctgacc 540 ctgtccaaag ctgattacga gaagcacaaa gtgtatgcat gcgaagtcac ccatcagggg 600 ctgtctagtc ccgtgacaaa gagctttaac cggggagagt gt 642 <210 > 11 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 11 gaggtgcagc tggtcgaatc tggaggagga ctggtgcagc caggagggtc actgagactg 60 agctgcgccg cttccggctt caacatcaag gagtactata tgcactgggt gaggcaggca 120 cctggcaaag gactggagtg ggtgggactg atcgacccag aacaggggaa caccatctac 180 gaccctaagt ttcaggatag ggcaaccatt tctgccgaca acagtaaaaa tacagcttat 240 ctgcagatga acagcctgag ggctgaagat actgcagtgt actattgcgc acgcgacacc 300 gcagcctact tcgattattg gggacagggc accctggt ca cagtgagctc cgcatcaact 360 aagggaccca gcgtgtttcc actggccccc tctagtaaat ccacttctgg aggcaccgct 420 gcactgggct gtctggtgaa ggattacttc ccagagcccg tcacagtgag ctggaactcc 480 ggggccctga ccagcggagt ccatacattt cctgctgtgc tgcagtcaag cgggctgtac 540 tccctgtcct ctgtggtcac cgtgccaagt tcaagcctgg gaactcagac ctatatctgc 600 aacgtgaatc acaagccttc aaatacaaaa gtcgacaaga aagtggaacc aaagagctgt 660 gataaaacac atacttgccc accttgtcct gcaccagagc tgctgggagg accaagcgtg 720 ttcctgtttc cacccaagcc caaagacacc ctgatgattt cccgcacacc agaagtcact 780 tgcgtggtcg tggacgtgtc tcacgaggac cccgaagtca agttcaactg gtacgtggat 840 ggcgtcgagg tgcataatgc caagacaaaa ccccgggagg aacagtacaa ctccacatat 900 agagtcgtgt ctgtcctgac tgtgctgcac caggactggc tgaacgggaa ggagtataag 960 tgcaaagtga gtaataaggc cctgcccgct cctatcgaga aaacaattag caaggccaaa 1020 ggccagcctc gagaaccaca ggtgtacact ctgcctccat ctcgggacga gctgactaag 1080 aaccaggtca gtctgacctg tctggtgaaa ggattctatc ccagcgatat cgctgtggag 1140 tgggaatcca atggccagcc tgagaacaat tacaagacca caccccctgt gct ggactct 1200 gatggcagtt tctttctgta tagtaagctg accgtcgata aatcacgatg gcagcagggg 1260 aacgtgttca gctgttcagt gatgcacgaa gccctgcaca accattacac ccagaagagc 1320 ctgagcctgt ctcccggc 1338 <210> 12 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide < 400> 12 gacatccaga tgacacagtc ccctagctcc ctgagtgcct cagtggggga cagagtcact 60 atcacctgcc gggcttccag agatattaag tcttacctga actggtatca gcagaagcca 120 ggcaaagcac ccaaggtgct gatctactat gccaccagtc tggctgaagg agtgccttca 180 cggttcagcg gctccgggtc tggaactgac tacacactga ctatttctag tctgcagcct 240 gaggatttcg ctacctacta ttgcctgcag cacggcgaat ccccatggac ttttggccag 300 gggaccaaag tggagatcaa gaggacagtg gccgctccat ccgtcttcat ttttccccct 360 tctgacgaac agctgaaatc aggaactgcc agcgtggtct gtctgctgaa caatttctac 420 ccccgcgagg caaaagtgca gtggaaggtc gataacgccc tgcagagtgg caattcacag 480 gagagcgtga cagaacagga ctccaaagat tctacttata gtctgtcaag caccctgaca 540 ctgtctaagg ctgattacga gaagcacaaa gtgtatgcat gcgaagtcac ccatcagggg 600ctgtcctctc ccgtgacaaa gagctttaat cggggagagt gt 642

Claims (30)

A multispecific or bispecific antigen-binding polypeptide construct comprising the structure H1-L1:H2-L2 comprising at least a first heterodimer (H1-L1) and a second heterodimer (H2-L2),
The first heterodimer (H1-L1) comprises a first human or humanized immunoglobulin G heavy chain polypeptide sequence (H1) and a first human or humanized immunoglobulin G light chain polypeptide sequence (L1), and comprises a first epitope has a first Fab region that binds; The second heterodimer (H2-L2) comprises a second human or humanized immunoglobulin G heavy chain polypeptide sequence (H2) and a second human or humanized immunoglobulin G light chain polypeptide sequence (L2), and has a second epitope has a second Fab region that binds; here
at least one of the H1 sequence or L1 sequence of the first heterodimer is distinct from the corresponding H2 sequence 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);
L1 and L2 each comprise at least a light chain variable domain (V _L domain) and a light chain constant domain ( _CL domain);
H1, H2, L1, and L2 each contain one or more amino acid modifications, wherein H1 preferentially pairs with L1 over L2 to form H1-L1, or H2 preferentially pairs with L2 over L1. to form H2-L2, or both;
The thermal stability, as measured by the melting temperature (Tm) determined by differential scanning calorimetry of the first and second Fab regions of the first and second heterodimers, was determined by the corresponding heterodimers without amino acid modifications. is within 0, 1, or 1.5° C. of the Tm of the monomer;
A construct wherein H1, L1, H2 and L2 contain one or more of the following amino acid modifications at positions identified according to the Kabat numbering system:
a) H1 comprising amino acid substitutions 124R and 172T, L1 comprising amino acid substitutions 133G, 174R and 176D, H2 comprising amino acid substitutions 124E and 172R, and L2 comprising amino acid substitutions 133G and 176R;
b) H1 comprising amino acid substitutions 139W, 143E, 145T and 179E, L1 comprising amino acid substitutions 116A, 124R, 135V and 178R, H2 comprising amino acid substitution 179K, and amino acid substitutions 124E, 135W, 160E and 180E. to L2;
c) H1 comprising amino acid substitutions 124A, 143F and 179K, L1 comprising amino acid substitutions 124E, 133W, 176T, 178L and 180E, H2 comprising amino acid substitutions 124W, 143E, 145T and 179E, and amino acid substitutions 124R, 133A , L2 including 176T and 178R;
d) H1 comprising amino acid substitutions 143E, 145T and 179E, L1 comprising amino acid substitutions 124R and 178R, H2 comprising amino acid substitution 143R, and L2 comprising amino acid substitutions 124E and 133E;
e) H1 comprising amino acid substitutions 143E, 145T and 179E, L1 comprising amino acid substitutions 124R and 178R, H2 comprising amino acid substitution 179R, and L2 comprising amino acid substitutions 124E, 178E and 180E;
f) H1 comprising amino acid substitutions 143E, 145T and 179E, L1 comprising amino acid substitutions 124R and 178R, H2 comprising amino acid substitution 179K, and L2 comprising amino acid substitutions 124E and 180E; 2. The method of claim 1, wherein H1 comprises amino acid substitutions 124R and 172T, L1 comprises amino acid substitutions 133G, 174R and 176D, H2 comprises amino acid substitutions 124E and 172R, and L2 comprises amino acid substitutions 133G and 176R. The construct, which is to do. 2. The method of claim 1, wherein H1 comprises amino acid substitutions 139W, 143E, 145T and 179E, L1 comprises amino acid substitutions 116A, 124R, 135V and 178R, H2 comprises amino acid substitution 179K, and L2 comprises amino acid substitution 124E. , 135W, 160E and 180E. 2. The method of claim 1, wherein H1 comprises amino acid substitutions 124A, 143F and 179K, L1 comprises amino acid substitutions 124E, 133W, 176T, 178L and 180E, H2 comprises amino acid substitutions 124W, 143E, 145T and 179E , wherein L2 comprises the amino acid substitutions 124R, 133A, 176T and 178R. 2. The method of claim 1, wherein H1 comprises amino acid substitutions 143E, 145T and 179E, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 143R and L2 comprises amino acid substitutions 124E and 133E. phosphorus, construct. 2. The method of claim 1, wherein H1 comprises amino acid substitutions 143E, 145T and 179E, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitution 179R and L2 comprises amino acid substitutions 124E, 178E and 180E. The construct, which is to do. 2. The method of claim 1, wherein H1 comprises amino acid substitutions 143E, 145T and 179E, L1 comprises amino acid substitutions 124R and 178R, H2 comprises amino acid substitutions 179K and L2 comprises amino acid substitutions 124E and 180E. phosphorus, construct. 8. The construct of any one of claims 1-7, wherein L1 and L2 are kappa light chains. 8. The method according to any one of claims 1 to 7, wherein H1, H2, L1 and L2 are co-expressed in cells or mammalian cells, or H1, H2, L1 and L2 are co-expressed 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 pairs with L1 over L2, and H2 with L1. to preferentially pair with L2. 8. The method according to any one of claims 1 to 7, wherein the pairing of modified H1-L1 or H2-L2 heterodimer pairs occurs in the corresponding H1-L1 or H2-L2 heterodimer pairs without amino acid modifications. Constructs that are larger than each pairing observed. 8. The method according to any one of claims 1 to 7, wherein the affinity of each heterodimer for an antigen to which each heterodimer binds, as measured by surface plasmon resonance (SPR) or FACS, to the same antigen. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, or 50 times the affinity of each unmodified heterodimer for , construct. 8. The method of any one of claims 1-7, wherein the construct further comprises an Fc comprising a first CH3 sequence and a second CH3 sequence, wherein the first CH3 sequence is present or absent one or more linkers. and wherein the second CH3 sequence is associated with the second heterodimer with or without one or more linkers. 13. The construct of claim 12, wherein 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. . 13. The construct of claim 12, wherein Fc is a heterodimeric Fc. 15. The construct of claim 14, wherein the Fc comprises one or more modifications in at least one of the CH3 sequences. 16. The construct of claim 15, wherein the Fc comprises one or more modifications in at least one of the CH3 sequences that promote formation of a heterodimeric Fc with stability comparable to wild-type homodimeric Fc. 17. The method of claim 16, wherein Fc is
i) a heterodimeric IgG1 Fc with the modification L351Y_F405A_Y407V in a first Fc polypeptide and the modification T366L_K392M_T394W in a second Fc polypeptide;
ii) a heterodimeric IgG1 Fc with the modification L351Y_F405A_Y407V in the first Fc polypeptide and the modification T366L_K392L_T394W in the second Fc polypeptide;
iii) a heterodimeric IgG1 Fc with the modification T350V_L351Y_F405A_Y407V in the first Fc polypeptide and the modification T350V_T366L_K392L_T394W in the second Fc polypeptide;
iv) a heterodimeric IgG1 Fc with modifications T350V_L351Y_F405A_Y407V in a first Fc polypeptide and T350V_T366L_K392M_T394W in a second Fc polypeptide; or
v) a heterodimeric IgG1 Fc with the modification T350V_L351Y_S400E_F405A_Y407V in a first Fc polypeptide and the modification T350V_T366L_N390R_K392M_T394W in a second Fc polypeptide
wherein the numbering of residues in the Fc polypeptide follows the EU numbering system. 13. The construct of claim 12, wherein the Fc further comprises at least one CH2 sequence. 19. The construct of claim 18, wherein the CH2 sequence(s) of Fc comprises one or more modifications. 13. The construct of claim 12, wherein the Fc comprises one or more modifications that promote selective binding of Fc-gamma receptors. 13. The construct of claim 12, wherein Fc is linked to the heterodimer by one or more linkers, or Fc is linked to H1 and H2 by one or more linkers, optionally wherein the one or more linkers are one or more polypeptide linkers. 22. The construct of claim 21, wherein the one or more linkers comprise one or more antibody hinge regions, optionally wherein the one or more linkers comprise one or more IgG1 hinge regions. 23. The construct of claim 22, wherein one or more linkers comprise one or more modifications. 24. The construct of claim 23, wherein the one or more modifications promote selective binding of Fc-gamma receptors. 13. The construct of claim 12, which is conjugated to a therapeutic or drug moiety. 8. An isolated polynucleotide or set of isolated polynucleotides comprising at least one sequence encoding the construct of any one of claims 1-7. A polynucleotide comprising at least one of the polynucleotide or set of polynucleotides of claim 26, optionally from the group consisting of a plasmid, a multi-cistronic vector, a viral vector, a non-episomal mammalian vector, an expression vector and a recombinant expression vector. A vector or set of vectors, to be selected. An isolated cell comprising the polynucleotide or set of polynucleotides of claim 26 or the vector or set of vectors of claim 27 and optionally being a yeast cell, bacterial cell, hybridoma, Chinese Hamster Ovary (CHO) cell or HEK293 cell. comprising the construct of any one of claims 1 to 7 and a pharmaceutically acceptable carrier, optionally buffers, antioxidants, low molecular weight molecules, drugs, proteins, amino acids, carbohydrates, lipids, chelating agents, stabilizers and A pharmaceutical composition for the treatment of a disease or disorder, further comprising one or more substances selected from the group consisting of excipients. A method of obtaining the construct of any one of claims 1 to 7 from a host cell culture,
(a) obtaining a host cell culture comprising at least one host cell comprising one or more nucleic acid sequences encoding said construct; and
(b) recovering the construct from the host cell culture.

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