WO2014014796A1 - Multi-specific igg-(fab)2 constructs containing t-cell receptor constant domains - Google Patents

Multi-specific igg-(fab)2 constructs containing t-cell receptor constant domains Download PDF

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WO2014014796A1
WO2014014796A1 PCT/US2013/050436 US2013050436W WO2014014796A1 WO 2014014796 A1 WO2014014796 A1 WO 2014014796A1 US 2013050436 W US2013050436 W US 2013050436W WO 2014014796 A1 WO2014014796 A1 WO 2014014796A1
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seq
antibody
compound
igg
fab
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Stephen John DEMAREST
Xiufeng Wu
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Eli Lilly and Co
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Eli Lilly and Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present disclosure relates to IgG-(Fab)2 multi-specific compounds containing
  • T-Cell receptor constant domains and methods of making such multi-specific compounds.
  • Therapeutic antibodies allow for the modulation of many disease conditions which provides benefits to patients. There are instances in which modulating more than one target could provide even greater benefit to patients. Although administration of two therapeutic antibodies is possible, practical considerations such as non-compatibility of formulations when combining multiple therapeutic antibodies into a single dosage form, and increased injections when administering multiple therapeutic antibodies as separate agents, may limit the possible benefit. A multi-specific compound could help minimize these concerns, and may also provide biological activities distinct from the combined administration of individual agents.
  • IgG- scFv One of the most commonly used multi-specific compound platforms is the IgG- scFv.
  • an scFv is linked to each end of the heavy or light chain of an antibody.
  • the IgG-scFv platform attempts to combine the activities of two antibodies while maintaining IgG-like pharmacokinetics and immune effector function.
  • the VJI and VL domains can exhibit attenuated stability and the exposure of hydrophobic surface area due to weaker HC/LC association can lead to aggregation.
  • IgG-scFv multispecific compounds To make IgG-scFv multispecific compounds viable, the biophysical aspects of the scFv within each particular IgG-scFv must be engineered to overcome expression, folding, affinity, solubility, and stability problems. Engineering scFvs to sufficiently overcome these issues is not straightforward, requiring significant time and resources for every multi-specific compound. As such, the IgG-scFv configuration may not always be a viable option for generating a multi-specific compound.
  • HC and LC association are strong, with limited interdomain dynamics.
  • manufacturing an antibody with a Fab linked to each HC or LC of the antibody (IgG-(Fab) 2 ) compound is challenging because the two LCs of an IgG-(Fab) 2 compound will bind heterogeneously to the two HC Fd (i.e., VJI and C jj l) regions within the compound generating unacceptable heterogeneity
  • the compounds of the present invention achieve specific assembly of IgG-(Fab) 2 compounds by replacing the Fab or antibody constant domains (CJJI/CL) with T-cell receptor (TCR) constant domains.
  • the present disclosure provides that the IgG-(Fab) 2 of the present invention contain the TCR a constant domain in the HC polypeptide to allow for proper assembly of the IgG-(Fab) 2 such that the specificities of the Fab and the antibody are maintained.
  • the compounds of the present invention allow for the specificities and binding activities of the variable regions of two therapeutic antibodies to be combined in one compound.
  • the present disclosure provides a compound comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, in which a. the first polypeptide chain has the formula VLi - C , wherein i. VLi is a first antibody light chain variable region, and
  • C is a T-Cell Receptor ⁇ constant domain; and b. the second polypeptide has the formula Vl ?- CL, wherein
  • VL2 is a second antibody light chain variable region
  • ii. CL is a light chain ⁇ or ⁇ constant domain
  • the third polypeptide chain has a formula selected from the ; consisting of
  • VHi is a first antibody heavy chain variable region
  • Ca is a T-Cell Receptor a constant domain
  • Xi is a peptide linker or is absent
  • Cfj2 ⁇ Cfj3 are heavy chain constant domains 2 and 3, and
  • H is a hinge region between CHI and Cm
  • X2 is a peptide linker or is absent
  • VH2 is a second antibody heavy chain variable region
  • Cjil is a heavy chain constant domain 1
  • first and second polypeptide chains associate with the third polypeptide chain to form a binding site (VL1-VH1) for a first binding partner and a binding site for a second binding partner (VL2-VH2), respectively.
  • the binding partners may be distinct antigens or proteins, or may be different positions on an antigen or protein.
  • the present disclosure also provides a mammalian cell containing DNA encoding three polypeptides,
  • a first polypeptide has a formula the first polypeptide chain has the formula VI - Cp, wherein
  • VLi is a first antibody light chain variable region
  • the second polypeptide has the formula Vl ?- CL, wherein
  • VL2 is a second antibody light chain variable region
  • CL is a light chain ⁇ or ⁇ constant domain; and c. the third polypeptide has a formula selected from the group consisting of
  • VHi is a first antibody heavy chain variable region
  • Ca is a T-Cell Receptor a constant domain
  • Xi is a peptide linker or is absent
  • Cfj2 ⁇ Cfj3 are heavy chain constant domains 2 and 3, and
  • H is a hinge region between C HI and C H2 .
  • X2 is a peptide linker or is absent
  • VH2 is a second antibody heavy chain variable region
  • Cm is a heavy chain constant domain 1.
  • the present disclosure provides a compound comprising an antibody and two Fabs, wherein the V H containing polypeptides of the Fab are linked to the heavy chains of the antibody and wherein the C HI constant regions of the antibody or Fab fragments are replaced with T-cell receptor a constant domains (Ca) and the corresponding light chain constant domains of the antibody or Fabs are replaced with T-cell receptor ⁇ constant domains (C ) such that the Ca and C domains allow the corresponding variable regions to associate to form an antigen binding fragment and the C HI and light chain constant regions allow the variable regions associate to form another antigen binding fragment.
  • the present disclosure provides a compound comprising an antibody and two Fabs, wherein the V H containing polypeptides of the Fab are linked to the heavy chains of the antibody and wherein the C HI regions of the Fab fragments are replaced with T-cell receptor a constant domains (Ca) and the constant domains of the Fab light chains are replaced with T-cell receptor ⁇ constant domains (C ) such that the Ca and C domains associate to form part of an antigen binding fragment.
  • the present disclosure also provides a compound comprising an antibody and two Fabs, wherein the V H containing polypeptides of the Fab are linked to the heavy chains of the antibody and wherein the C H I regions of the antibody are replaced with T-cell receptor a constant domains (Ca) and the constant domains of the antibody light chain are replaced with T-cell receptor ⁇ constant domains (C ) such that the Ca and C domains associate to form part of an antigen binding fragment.
  • the present disclosure provides a compound comprising three polypeptide chains, in which
  • the first polypeptide chain comprises the formula VHi- Ca wherein
  • VHi is a first antibody heavy chain variable region
  • Ca is a T-cell receptor a constant domain
  • the second polypeptide chain comprises the formula VH 2 -CHI-H-CH 2 -CH3 wherein
  • V3 ⁇ 4 is a second antibody heavy chain variable region, and CHI, CH 2 , and CH3 are heavy chain constant domains 1 , 2, and 3,
  • H is a hinge region
  • the third polypeptide chain comprises the formula VLi-Cp-Xi-VL 2 -CL,
  • VLi is a first antibody light chain variable region
  • CP is a T-Cell Receptor ⁇ constant domain
  • Xi is a linker which can be present or absent
  • VL 2 is a second antibody light chain region
  • CL is either a C k or light chain antibody constant domain, in which the first and second polypeptide chains associate with the third polypeptide chain to form a binding site (VLi-VHi) for a first binding partner and a binding site for a second binding partner (VL 2 -VH 2 ).
  • the present disclosure also provides a compound comprising three polypeptide chains, wherein
  • a first polypeptide chain has the formula VHI-CHI-H-CH 2 -CH3,
  • VHi is a first antibody heavy chain variable region
  • CHI, CH 2 , and CH3 are heavy chain constant domains 1 , 2, and 3,
  • H is a hinge region
  • a second polypeptide chain has the formula VL 1 -CL-VL 2 - Cp, wherein
  • VLi is a first antibody light chain variable region
  • CL is either C k or C ⁇
  • VL 2 is a second light chain variable region
  • CP is a T-Cell Receptor ⁇ constant domain
  • the third polypeptide has the formula VH 2 - Ca, wherein
  • VH 2 is a second antibody heavy chain variable region and Ca is a T-Cell Receptor a constant domain,
  • first and third polypeptide chains associate with the second polypeptide chain to form a binding site (VL1-VH1) for a first binding partner and a binding site for a second binding partner (VL 2 -VH 2 ).
  • the present disclosure also provides a compound comprising three polypeptide chains, wherein
  • a first polypeptide chain has the formula VLi-CL, wherein
  • VLi is a first antibody light chain variable region
  • CL is either C k or Cx
  • a second polypeptide chain has the formula VL 2 - Cp, wherein
  • VL 2 is a second antibody light chain variable region
  • CP is a T-Cell Receptor ⁇ constant domain
  • a third polypeptide chain has the formula VH I -C HI -X I -VH 2 - Ca, wherein
  • VHi is a first antibody heavy chain variable region
  • C HI is a heavy chain constant domain 1 .
  • Xi is a linker
  • VH 2 is a second antibody heavy chain variable region
  • Ca is a T-Cell Receptor a constant domain
  • first and second polypeptide chains associate with the third polypeptide chain to form a binding site (VHi-VLi) for a first binding partner and a binding site for a second binding partner (VH 2 -VL 2 ).
  • the present disclosure also provides a compound comprising three polypeptide chains, wherein
  • a first polypeptide chain has the formula VLi- Cp, wherein
  • VLi is a first antibody light chain variable region
  • CP is a T-Cell Receptor ⁇ constant domain
  • a second polypeptide has the formula VL 2 -CL, wherein
  • VL 2 is a second antibody light chain variable region, and CL is either C k or Cx, and c) a third polypeptide has the formula VHi- Ca-Xi-VH2-C , wherein
  • VHi is a first antibody heavy chain variable region
  • Ca is a T-Cell Receptor a constant domain
  • Xi is a linker
  • VH 2 is a second antibody heavy chain variable
  • CHI heavy chain constant domain 1
  • first and second polypeptide chains associate with the third polypeptide chain to form a binding site (VHi-VLi) for a first binding partner and a binding site for a second binding partner (VH2-VL2).
  • the present invention also provides a process for producing any of the proceeding compounds comprising cultivating the mammalian cells containing DNA encoding a compound of the present invention under conditions such that the compound is expressed and recovering the compound.
  • Fig. 1 Schematic diagram of Format #1 of a TCR-based IgG-(Fab) 2 compound, comprising three polypeptide chains: VI - C , VL 2 -CL, and (VHI-C(X)-X1-(CH 2 -CH3)-
  • VLi is a first antibody light chain variable region
  • CP is a T- Cell Receptor ⁇ constant domain
  • VL2 is a second antibody light chain variable region
  • CL is a light chain ⁇ or ⁇ constant domain
  • VHi is a first antibody heavy chain variable region
  • Ca is a T-Cell Receptor a constant domain
  • XI is a peptide linker or is absent
  • CH 2 -CH3 are heavy chain constant domains 2 and 3
  • X2 is a peptide linker or is absent
  • VH2 is a second antibody heavy chain variable region
  • CHI is a heavy chain constant domain 1.
  • Fig. 2 Schematic diagram of Format #2 of a TCR-based IgG-(Fab) 2 compound, comprising three polypeptide chains: VI - C , VL 2 -CL, and (VH 2 -CHI)-(CH 2 -CH3)- X2-(VH ! -Ca).
  • Fig. 3 Schematic diagram of Format #3 of a TCR-based IgG-(Fab) 2 compound, comprising three polypeptide chains: VLi - C , VL 2 -CL, and (VHi-Ca)-
  • an "antibody” The general structure of an "antibody” is very well-known. For an antibody of the IgG type, there are four amino acid chains (two “heavy” chains and two “light” chains) that are cross-linked via intra- and inter-chain disulfide bonds. When expressed in certain biological systems, antibodies having unmodified human Fc sequences are glycosylated in the Fc region. Antibodies may be glycosylated at other positions as well. The subunit structures and three-dimensional configurations of antibodies are well known.
  • Each heavy chain is comprised of an N-terminal heavy chain variable region ("VH") and a heavy chain constant region ("CH")-
  • the heavy chain constant region is comprised of three domains (CHI, CH2, and CH3) for IgG as well as a hinge region ("hinge") between the CHI and CH 2 domains.
  • Each light chain is comprised of a light chain variable region ("VL”) and a light chain constant region (“CL”).
  • the CL may be of the kappa (" ⁇ ”) or lambda (“ ⁇ ”) isotypes.
  • variable regions of each light/heavy chain pair to form binding sites can be subdivided into regions of hypervariability, termed complementarity determining regions ("CDRs"), interspersed with regions that are more conserved, termed framework regions ("FR").
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • CDRH1 , CDRH2, and CDRH3 the 3 CDRs of the heavy chain
  • CDRL1 , CDRL2 and CDRL3 the 3 CDRs of the light chain
  • the CDRs contain most of the residues which form specific interactions with the antigen.
  • the assignment of amino acids to each domain is in accordance with well-known conventions [e.g., Kabat, "Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)].
  • An antibody may be derived from a single copy or clone (monoclonal antibody (mAb)), including e.g., any eukaryotic, prokaryotic, or phage clone.
  • mAb monoclonal antibody
  • an antibody of the present invention exists in a homogeneous or substantially homogeneous population of antibody molecules.
  • a full-length antibody comprises full length or substantially full length constant regions, including the Fc region.
  • An "antigen-binding fragment" of such an antibody is any shortened form of a full length antibody that comprises the antigen-binding variable regions and retains antigen-binding capability.
  • Such shortened forms include, e.g., a Fab fragment, Fab' fragment or F(ab') 2 fragment that includes the CDRs or the variable regions of the antibodies disclosed.
  • An antibody of the present invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art.
  • An antibody of the present invention is an engineered antibody that has been engineered to comprise framework, hinge, or constant regions derived from fully human frameworks, hinge, or constant regions containing one or more amino acid substitutions, deletions, or additions therein. Further, an antibody of the present invention is substantially non-immunogenic in humans.
  • the framework regions of an antibody of the present invention are of human origin or substantially human (at least 95%, 97% or 99% of human origin.)
  • the sequences of framework regions of human origin may be obtained from The Immunoglobulin
  • T-cell receptors are used to recognize foreign antigenic peptides displayed by various antigen presenting cells of the immune system. Binding of T-cells to antigen- presenting cells occurs via interactions between major histocompatability complex
  • MHC antigen-presenting cells
  • TCRs T- cell receptors
  • Formation of a cell-bridging MHC- peptide-TCR complex typically results in stimulation and activation of the T-cells, a primary step of an adaptive immune response.
  • TCRs are related to immunoglobulins in that they contain hypervariable V-class Ig-fold domains capable of generating an enormous diversity that enables them to recognize diverse peptidic antigens displayed by MHCs. Like antibody Fabs, they also contain C-class Ig-fold constant domains that help stabilize the TCR complex.
  • TCR constant domains have very different primary sequences than immunoglobulin Fab constant domains (-20% identity between the TCR a- and ⁇ -domains and IgG CHI and CL constant domains). Additional information regarding T-cell receptors and their constant domains may be found in The T Cell Receptor Factsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001, ISBN 0124413528.
  • Example 1- Certain Orientations of TCR Constant Regions May Substitute for CH1/CL Regions; Variable Region-Constant Region Linker Length
  • a- and ⁇ -constant domains (Cot and C , respectively) to replace the CHI of the heavy chain and light chain constant (CL) domains
  • varying constructs in a mammalian expression vector for transient expression in HEK293 cells are generated. Both orientations with the Cot or CP domains replacing the CHI domain or the CL domain are constructed.
  • this chimeric antibody format is called IgG-TCR.
  • An anti-human IL-17 IgG4/K (HC of SEQ ID NO:78 and LC of SEQ ID NO:79) antibody is utilized for generating IgG-TCR constructs.
  • CHI/ CL heterodimeric unit of the antibody is replaced using both Cot/CP and Cp/Cot orientations and varying V-gene/TCR constant domain linker sequences.
  • Each heavy chain (“HC") construct is co-transfected with each light chain (“LC”) construct to determine whether any format will allow for assembly, binding, and stability.
  • the linking regions between IgG V-genes and their constant regions are shorter than the linking regions between TCR V-genes and their respective constant regions.
  • the linker regions between the antibody V-genes and the TCR constant domains, as well as truncated versions of the a- and ⁇ -constant connector regions are generated within the IgG-TCR format.
  • the sequences of the constructs with truncations are denoted in Table 1.
  • Heavy chain genes are subcloned into a vector containing the gene sequence coding for a human IgG4 constant region resulting in the fusion products of the antibody V-genes and the TCR Cot and C genes with the IgG4PAA hinge-Fc domain.
  • Light chains are also subcloned into a vector.
  • the vector contains a common mouse antibody LC signal sequence that is translated in-frame as part of the expressed protein and cleaved prior to secretion.
  • the ligation mixture was used to transform E. coli strain TOP 10 competent cells (Invitrogen Corporation, Carlsbad, CA). Sequences are confirmed by DNA sequencing. Plasmid DNAs are used to transfect 293F cells for transient production of antibody protein.
  • SEQ ID NO:2 The sequence given by SEQ ID NO:2 represents a truncation of N-terminal Pro-
  • PN Asn (PN) from the native human Ca sequence (given by SEQ ID NO: l).
  • SEQ ID NO: 11 represents a truncation of N-terminal Glu-Asp-Leu-Asn (EDLN) from the native human CP sequence (given by SEQ ID NO: 10).
  • EDLN N-terminal Glu-Asp-Leu-Asn
  • SEQ ID NO: 10 The sequences of the variable regions, hinge, CH2, and CH3 are identical for these constructs. The only variation is in the CHi/CL domains.
  • a vector harboring the LC/TCR DNA sequence and a vector harboring the HC/TCR DNA sequence are transfected (1 :2 plasmid ratio for the HC and LC plasmids) into 293F cells using FreeStyleTM transfection reagents (Life Technologies). Transfected cells are grown at 37 °C in a 5% C0 2 incubator while shaking at 125 rpm. Secreted protein is harvested by centrifugation at >10,000 rpm for 5 min.
  • IgG-TCR is evaluated by SDS-PAGE. Approximately 5 ⁇ g protein is loaded in each well of Novex ® 4-20% Tris Glycine gels or 3-8% Tris-Acetate gels according to manufacturer protocols (Life Technologies). For reduced samples, 10% 0.5 M DTT in H 2 0 is added to the same prior to loading.
  • Analytical size exclusion chromatography (SEC) with in-line light scattering (SEC/LS) is performed for each sample. 30-80 ⁇ L ⁇ of each sample (-0.2-0.8 mg/mL) are injected onto a Sepax Zenix SEC 200 analytical HPLC (7.8x300 mm) column equilibrated in 10 mM phosphate, 150 mM NaCl, 0.02% NaN 3 , pH 6.8, using an Agilent 1100 HPLC system (Agilent Technologies). Static light scattering data for material eluted from the SEC column are collected using a miniDAWN TREOS static light scattering detector coupled to an Optilab T-rEX in-line refractive index meter (Wyatt Technologies).
  • UV data are analyzed using HPCHEM (Agilent). Molecular weights of the complexes are determined by their static light scattering profiles using ASTRA V (Wyatt Technologies). The SEC results (Table 2) demonstrate that the constructs aggregate to a similar level as a control IgG4 antibody.
  • kinetic surface Plasmon resonance (SPR) experiments are performed using a Biacore 3000 (GE Healthcare).
  • IgG-TCR proteins are captured onto CM5 sensorchip surfaces with an immobilized goat anti-human IgG-Fc polyclonal antibody (Jackson ImmunoResearch, Cat. #109-005-098).
  • the goat polyclonal antibody immobilization is achieved by injecting the protein (at 50 ⁇ g/mL, pH 5) over an NHS/EDC activated sensorchip surface followed by blocking with ethanolamine (as described by
  • the IgG or IgG-TCRs are captured onto the anti-human Fc sensorchip surface by injecting 10 ⁇ L ⁇ of protein at 0.1 and 0.5 mg/mL using a 2 ⁇ / ⁇ flow rate. Flow rates are increased to 10 ⁇ / ⁇ followed by secondary 30 ⁇ L ⁇ injections of IL-17 (SEQ ID NO:80) at 10 and 50 nM. Following a 15 minute dissociation period, the flow rate is increased to 60 ⁇ /min and sensorchip surfaces are regenerated using two consecutive 10 ⁇ injections of 0.1 M glycine, pH 2.0.
  • the experiment measures the percent activity by relating the amount of captured IgG or IgG-TCR (based on resonance units) with the secondary response generated using a saturating level of IL-17.
  • the percent activity of each IgG-TCR is calculated based on a comparison with the WT IgG:
  • RUjgQ X R an d RUigQ are the resonance units of the captured IgG-TCR or IgG, respectively
  • RUJL_I ⁇ is the saturating level of resonance units generated by the binding of IL-17 to the pre-loaded anti-IL-17 IgG or IgG-TCR.
  • TCR3 which had the Cot- and CP domains replacing the CL and CHI domains, respectively, demonstrated little activity in the assay indicating a near complete lack of LC/HC assembly (Table 2).
  • TCR2, TCR4, TCR6, and TCR8 (with the a- and ⁇ -constant regions replacing the CHI and CL domains, respectively) all had activity. (Table 2).
  • Table 2 Biochemical characterization of TCR1-TCR8.
  • CL domain of an immunoglobulin with C allows for the native-like heterotetramer formation of an IgG
  • Antibody I (HC SEQ ID NO. 34; LC SEQ ID NO. 33, trastuzumab), which binds to HER2, is used for the following experiments.
  • the variable regions of Antibody I are appended to the N-termini of TCR4 above.
  • the IgG-TCR construct containing the Antibody I Fv is denoted as tTCR-G4(+) and has HC and LC sequences of SEQ ID NO:36 and SEQ ID NO:35, respectively.
  • IgG4 hinge (SEQ ID NO:5) of tTCR- G4 is replaced with an IgGl hinge (SEQ ID NO: 6) resulting in tTCR-Gl(+) which has a HC of SEQ ID NO:37 and a LC of SEQ ID NO:35.
  • truncations are made in the C-terminus of ⁇ .
  • a new construct, with four residues truncated from the C-terminal sequence of C is created (SEQ ID NO: 12).
  • a construct tTCR-Gl(-) contains the same tTCR-Gl HC (SEQ ID NO: 37) with the modified tTCR-Gl LC (SEQ ID NO:38) truncated at its C-terminus.
  • pTCR-Gl(-) containing the Antibody II (HC SEQ ID NO:42; LC SEQ ID NO:41) variable regions is constructed.
  • the equilibrium dissociation constant, K D , and binding stoichiometry, n (used to determine the assembly), between the IgG-TCRs and hHER-2-Fc are determined using the linear relationship between [R]p and linear slope of RU/time (known as the velocity or Vi) within the first 80 seconds of the experiment:
  • [IgG_TCR] T total IgG_TCR concentration.
  • Table 3 -tTCR-Gl(+), tTCR-Gl(-), and pTCR-Gl(-) construct compositions.
  • C H2 -C H3 was that of SEQ ID NO: 15, except that the terminal Lys (K) was deleted.
  • n.d denotes not determined. Truncation of the C-terminal four amino acids of C results in a 5-fold average increase in protein expression as determined according to the procedure in Example 2 (data not shown). The proteins with the LC-C truncation are more uniformly the expected molecular weight based on in-line light scattering measurements and do not appear to be incompletely assembled. Additionally, truncating four LC-C -terminal amino acids eliminates apparent proteolysis, mis-assembly, or protein degradation that had been observed as multiple absorbance peaks eluting for non-truncated molecules (data not shown).
  • tTCR-Gl compounds could not inhibit hHER-2-Fc from binding an Antibody II-labeled surface and the pTCR-Gl(-) compound could not inhibit hHER-2- Fc binding to an Antibody I-labeled surface, indicating that the specificity for their particular epitopes is intact.
  • Different batches of tTCR-Gl (+) appeared to give varying levels of assembly, as measured by the stoichiometry of hHER2-Fc blocking in the assay, perhaps because of differences in plasmid levels that were transfected (data not shown).
  • the tTCR-Gl(-) and pTCR-Gl(-) proteins consistently block binding of 20 nM hHER2- Fc to surfaces with Antibody I and Antibody II, respectively, at 20 nM concentrations indicating they are 100% assembled.
  • an IgGl hinge including the interchain cysteine that forms a disulfide with LC
  • the LC-C truncation results in fully assembled IgG-TCRs.
  • tTCR-Gl(-) and pTCR-Gl(-) HC plasmids are transfected with both their cognate C constant domain containing LCs and the natural (non-TCR-domain containing) LC plasmids (Table 5).
  • the wild-type IgGl Antibody I (trastuzumab) and Antibody II (pertuzumab) HC plasmids are transfected with their cognate natural LCs and their LCs containing C constant domains (Table 5).
  • An OctetRed (ForteBio) biosensor assay is used to evaluate the specificity of the optimized Antibody I and Antibody II IgG-TCR constructs within this Example.
  • the method includes the use of an anti-human IgG-Fc specific biosensor (ForteBio) to capture the IgG-TCR HC or wild-type IgG HC transfected in the presence of both the wild-type and TCR -containing LCs (Table 5) at 10 ⁇ g/mL in Octet buffer for 5 minutes.
  • the captured IgGs or IgG-TCRs are then used to capture an anti-kappa CL domain- specific murine mAb (Sigma-Aldrich Cat.
  • Table 5 Co-transfection of Antibody I and Antibody II IgG-TCR and IgG HCs with both natural LCs or LCs containing TCR- ⁇ constant domains.
  • Wild-type IgG HCs show strong selective binding to their cognate wild-type LC when expressed in the presence of a CP-containing LC with the same variable domains. Additionally, Coc-containing IgG HCs show selective binding to their cognate C - containing LCs when expressed in the presence of a wild-type LC.
  • IgG- (Fab) 2 To determine whether Coc and C domains can be used to generate compounds in which a Fab with one specificity is linked to an antibody with another specificity (IgG- (Fab) 2 ) and whether the HCs and LCs of such an IgG-(Fab) 2 can assemble appropriately to maintain their binding activities, IgG-(Fab) 2 with different Coc and C configurations are constructed.
  • the HC portion of the Fab is linked to the N- terminus of the HC of the antibody and in another configuration, the HC portion of the Fab is linked to the C-terminus of the antibody.
  • the CHI region of the antibody or Fab is replaced with a Coc domain, while the corresponding LC constant domain is replaced with C .
  • Each of the compounds contains a HC containing both a VH/ CHI domain and a VH/COC domain and two LCs, one containing a VL/CL pair and the other containing a Vi/C -constant domain pair.
  • the combinations of HCs and LCs that compose each compound are listed in Table 7 and 8 and illustrated in Figures 1-4. All constructs fall into one of four formats, as shown below:
  • C- or “N-” denotes an additional Fab region (either wild-type Fab or Fab containing TCR-constant domains) appended to the C- or N-terminus of the HC, respectively, “t” refers to Antibody I Fv (trastuzumab Fv), “p” refers to Antibody II (pertuzumab Fv), “Gl” denotes a wild-type Fab while “TCR” denotes a Fab containing TCR-constant domains. The order with which the Gl and TCR sequences are listed within each name indicates the order within the primary sequence that the Fab regions occur.
  • C-tTCRpGl indicates that a Fab of Antibody II (pertuzumab Fab), which has antibody CHI/CL domains, is linked to the C-terminal end of the HC of Antibody I (trastuzumab) whose CHI/CL domains are replaced by TCR Coc/C , respectively.
  • IgG-(Fab) 2 compound characterization including protein expression, protein purification, and in vitro biochemical are performed as described in Example 1.
  • the binding/assembly properties of the IgG-Fab compounds are determined using the SPR- based solution equilibrium methodology described in Example 2.
  • dProtein displayed varying HC/LC compositions based on SEC.
  • HER-2-positive NCI-N87 ATCC Cat. #CRL-5822
  • HER-2-positive NCI-N87 ATCC Cat. #CRL-5822
  • BT474 (ATCC Cat. #HTB-20) tumor cell lines are cultured according to the guidelines provided by the ATCC. For flow cytometry, cells (-75% confluent) are lifted from their culture flasks using cell dissociation buffer (Cat. #13151014 Life Technologies), counted, and plated in 96- well round bottom tissue culture plates at 0.5 x 10 6 cells per well.
  • Mouse Ig Gl-PE (BD), Mouse anti-EGFR-PE (BD), Mouse anti-Her-2/neu-PE (BD), and Mouse anti-Her-3/erbB3-PE (BD) all are used at 20 ⁇ 1/0.5 ⁇ 10 6 cells. All mAbs are diluted in flow cytometry buffer (Dulbecco PBS w/2% FBS & 0.05% sodium azide & 10% NGS) and incubated for 45 minutes. The cells are centrifuged at 1500 rpm for 5 minutes at 4 C and washed three times with flow cytometry buffer. After final wash the cells are resuspended in flow cytometry buffer containing propidium iodide (PI,
  • NCI- N87 or BT-474 cell lines are seeded on 96-well plates at 1 x 10 3 cells per well and precultured in RPMI-1640 medium containing 10% FBS overnight.
  • RPMI-1640 medium containing 10% FBS 100, 10, 1, and 0.1 nM solutions of each test compound (or combinations of 100, 10, 1, and 0.1 nM of each test compound) in RPMI-1640 medium containing 10% FBS are added to the cells.
  • BT-474(breast cancer) cell lines are seeded in 12- well culture plates at 2.5 x 10 5 cells per well and grown in RPMI-1640 medium containing 10% FBS overnight. The next day, cells are treated with 100 nM mAbs or IgG-(Fab) 2 for 24, 48 and 72 hours at 37°C in 10% FBS containing medium. Cell lysates are made using cell lysis buffer (MSD Cat. #R60TX-3), and protein concentrations are measured using BCA protein assay (Pierce).
  • Phospho-HER-2, Phospho-HER-3 and Phospho-EGFR in cell lysates are measured using a Phospho-HER-2, Phospho-HER-3 and Phospho-EGFR multiplex MSD kit (Meso Scale Discovery). Plates are loaded with 16 ⁇ g total protein in duplicate, and incubated for 2 hours at room temperature with shaking. Next, plates are washed and detection antibody is added and incubated for 2 hours at room temperature with shaking. Plates are read on a Sector Imager6000 (Meso Scale Discovery).
  • Table 10 Inhibition of BT474 breast and NCI-N87 gastric cancer cell lines by the IgG-(Fab) 2 compounds.
  • Antibody I trastuzumab
  • Antibody II pertuzumab individually inhibited FBS-mediated tumor cell growth of both the BT474 and NCI-N87 cell lines.
  • the combination of Antibody I and II resulted in an increase in anti-proliferative activity.
  • the individual IgG-TCR proteins, tTCR-Gl(-) and pTCR- Gl(-), and the combination of these proteins demonstrated significant decreases in antiproliferative activity presumably due to the different Fab/hinge dynamics introduced using the subtly different connecting regions within the IgG-TCR format.
  • Table 10 also shows that the various IgG-(Fab) 2 compounds possess a spectrum of activities on HER-2- mediated tumor cell growth ranging from antagonistic to highly-antagonistic with the N- pTCRtGl being even more inhibitory in the NCI-N87 cell line than the Antibody I and II combination.
  • Example 6- IgG-(Fab) 2 combining the specificities of the anti-HER-2 mAb pertuzumab and the anti-EGFR mAbs cetuximab and matuzumab.
  • IgG-(Fab) 2 compounds targeting EFGR and HER-2 are generated.
  • Different constructs containing a cetuximab Fab and wild type pertuzumab (with IgGl CHI domain and kappa CL domain) as well as two additional constructs containing a matuzumab Fab and wild-type pertuzumab (with IgGl CHI domain and kappa CL domain), each in the IgG-TCR format are generated.
  • Cetuximab is a chimeric mouse/human mAb directed against EGFR (HC of SEQ ID NO: 60 and LC of SEQ ID NO:59), while matuzumab is a humanized anti-EGFR mAb (HC of SEQ ID NO:69 and LC of SEQ ID NO:68).
  • the HCs and LCs that comprise the IgG-Fab are provided in Table 11 and 12 as are the sequences of the antibody controls and an IgG-TCR control, mTCR-Gl(-).
  • “c” refers to Antibody III (cetuximab Fv)
  • m refers to Antibody IV (matuzumab Fv) containing Fab. The remainder of the nomenclature is consistent with Example 4.
  • Table 11 Composition of the anti-HER-2/anti-EGFR IgG-Fabs and relevant controls
  • Table 12 HC and LC sequence composition of the anti-HER-2/anti-EGFR IgG- (Fab) 2 and controls
  • the construction, expression, purification, and biophysical characterization of the anti-HER-2/anti-EGFR IgG-(Fab) 2 compounds is performed as described in the Example 4.
  • the four IgG-(Fab) 2 compounds designed to recognize both EGFR and HER-2 are expressed and purified (by protein G magnetic beads only - no secondary purification steps) at the 2 mL and 10 mL scales.
  • an ELISA assay is run. Specifically, clear 96-well round bottom high binding microtiter plates (Greiner) are coated overnight at 2-8 °C with 50 ⁇ /well 1 ⁇ g/mL hEGFR-Fc (Cat. #344-ER-050, R&D systems) in a 50 mM Na 2 C0 3 pH 9.4 buffer. The plate is washed 4X times with PBST and blocked with 100 ⁇ /well casein buffer (Pierce) for 1 hr at 37 °C.
  • test compounds are added at 50 ⁇ /well and 30 ⁇ g/mL (20 nM) and serially diluted 1 :2 down the plate.
  • the test compounds are incubated on the plate for 1 hr at 37 °C.
  • the plate is washed 4X times with PBST and 50 ⁇ 7 well 0.2 ⁇ g/mL hHER-2-Histag (Sino Biologies) is added for 1 hr at 37 °C.
  • the plate is then washed 4X times with PBST followed by the addition of a 50 secondary anti-Histag-HRP antibody (PENTA-His-HRP, Qiagen) diluted 1 :1000 in PBST.
  • PENTA-His-HRP PENTA-His-HRP, Qiagen
  • the secondary antibody is incubated for 1 hr at 37 °C.
  • the plate is then washed 4X times with PBST and 100 ⁇ 1-component 3,3',5,5'- Tetramethylbenzidine (TMB) substrate is added (KPL laboratories). After approximately 10 minutes, 100 ⁇ /well 1% H 3 PO4 (in H 2 0) is added to quench the reaction. Absorbance (450 nm) of every well in the plate is read using a SpectraMax UV plate reader
  • Table 13 Characterization of TCR-based IgG-Fabs that recognize EGFR and HER-2.
  • the IgG-(Fab) 2 compounds have the correct molecular weight based on SDS-PAGE and analytical SEC and are primarily monodisperse with ⁇ 3- 10% soluble aggregates. Also, all four IgG-(Fab) 2 compounds are capable of binding both EGFR and HER-2 based on positive signals in the sandwich ELISA and the measured potencies are listed in Table 13.
  • the results of the expression and biophysical characterization indicate the IgG- (Fab) 2 compounds not only express at their expected molecular weights, but display relatively ideal starting biophysical properties.
  • the dual- specificity binding ELISA demonstrates the ability to bind both antigens for these IgG-Fab constructs.
  • IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP SEQ ID NO: 3 TCR a N-terminal truncated IgG4 Fc region
  • SEQ ID NO:25 anti-IL-17 LC Vi/Ccc
  • SEQ ID NO:27 anti-IL-17 HC V H /Cp/IgG4Fc
  • SEQ ID NO:33 Trastuzumab LC
  • IgG_TCR BsAb HC denoted C-tTCRpGl (C-terminal Fab fusion is wild- type pertuzumab Fab)
  • IgG_TCR BsAb HC denoted C-pGltTCR (C-terminal Fab fusion is IgG_TCR trastuzumab Fab)
  • SEQ ID NO:55 Trastuzumab VL/TCR-P LC
  • YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:77 mTCR_Gl(-) HC (IgG-TCR)

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Description

Multi- Specific IgG-(Fab)2 Constructs Containing T-Cell Receptor Constant
Domains
The present disclosure relates to IgG-(Fab)2 multi-specific compounds containing
T-Cell receptor constant domains and methods of making such multi-specific compounds.
Therapeutic antibodies allow for the modulation of many disease conditions which provides benefits to patients. There are instances in which modulating more than one target could provide even greater benefit to patients. Although administration of two therapeutic antibodies is possible, practical considerations such as non-compatibility of formulations when combining multiple therapeutic antibodies into a single dosage form, and increased injections when administering multiple therapeutic antibodies as separate agents, may limit the possible benefit. A multi-specific compound could help minimize these concerns, and may also provide biological activities distinct from the combined administration of individual agents.
One of the most commonly used multi-specific compound platforms is the IgG- scFv. In this platform, an scFv is linked to each end of the heavy or light chain of an antibody. The IgG-scFv platform attempts to combine the activities of two antibodies while maintaining IgG-like pharmacokinetics and immune effector function. In scFvs, the VJI and VL domains can exhibit attenuated stability and the exposure of hydrophobic surface area due to weaker HC/LC association can lead to aggregation.
To make IgG-scFv multispecific compounds viable, the biophysical aspects of the scFv within each particular IgG-scFv must be engineered to overcome expression, folding, affinity, solubility, and stability problems. Engineering scFvs to sufficiently overcome these issues is not straightforward, requiring significant time and resources for every multi-specific compound. As such, the IgG-scFv configuration may not always be a viable option for generating a multi-specific compound.
In Fabs, antibody heavy chain (HC) and light chain (LC) association is strong, with limited interdomain dynamics. However, manufacturing an antibody with a Fab linked to each HC or LC of the antibody (IgG-(Fab)2) compound is challenging because the two LCs of an IgG-(Fab)2 compound will bind heterogeneously to the two HC Fd (i.e., VJI and Cjjl) regions within the compound generating unacceptable heterogeneity
(sixteen HC/LC/LC pairings are possible). The compounds of the present invention achieve specific assembly of IgG-(Fab)2 compounds by replacing the Fab or antibody constant domains (CJJI/CL) with T-cell receptor (TCR) constant domains. In addition, the present disclosure provides that the IgG-(Fab)2 of the present invention contain the TCR a constant domain in the HC polypeptide to allow for proper assembly of the IgG-(Fab)2 such that the specificities of the Fab and the antibody are maintained. The compounds of the present invention allow for the specificities and binding activities of the variable regions of two therapeutic antibodies to be combined in one compound.
While chimeric proteins containing an antibody variable region linked to a TCR constant domain have been disclosed (Kuwana, et al., Biochemical and Biophysical Research Communications, Vol. 149, No. 3, 1987 and Seimiya, et al., Journal of Biochemistry 113, 687-691 , 1993), these references do not demonstrate appropriate assembly of a Vjj -Cot and a VL -C polypeptides in the context of an IgG-(Fab)2 as presently disclosed. Kuwana, et al. and Seimiya, et al. did not provide any disclosure or suggestion that using a TCR a constant domain in the heavy chain of an IgG-Fab, would allow for specific assembly of the polypeptide chains of an IgG-(Fab)2 compound such that the binding specificities of the Fab and the antibody would be maintained.
The present disclosure provides a compound comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, in which a. the first polypeptide chain has the formula VLi - C , wherein i. VLi is a first antibody light chain variable region, and
ii. C is a T-Cell Receptor β constant domain; and b. the second polypeptide has the formula Vl ?- CL, wherein
i. VL2 is a second antibody light chain variable region, and ii. CL is a light chain κ or λ constant domain; and the third polypeptide chain has a formula selected from the ; consisting of
(VH1-Ca)-X1-(CH2-CH3)-X2-(VH2 -CHl),
(VH2 CHl)-(H-CH2-CH3)-¾-(VHi-Ca),
(VH1 Ca)-X2-(VH2 -CHI)-(H-CH2-CH3)> and (VH2-CHl)-X2-(VH1-Ca)-X1-(CH2-CH3)'
wherein
VHi is a first antibody heavy chain variable region, and
Ca is a T-Cell Receptor a constant domain and
Xi is a peptide linker or is absent, and
Cfj2~Cfj3 are heavy chain constant domains 2 and 3, and
H is a hinge region between CHI and Cm, and
X2 is a peptide linker or is absent, and
VH2 is a second antibody heavy chain variable region and
Cjil is a heavy chain constant domain 1; and
in which the first and second polypeptide chains associate with the third polypeptide chain to form a binding site (VL1-VH1) for a first binding partner and a binding site for a second binding partner (VL2-VH2), respectively. The binding partners may be distinct antigens or proteins, or may be different positions on an antigen or protein.
The present disclosure also provides a mammalian cell containing DNA encoding three polypeptides,
a. wherein a first polypeptide has a formula the first polypeptide chain has the formula VI - Cp, wherein
VLi is a first antibody light chain variable region, and
is a T-Cell Receptor β constant domain;
b. the second polypeptide has the formula Vl ?- CL, wherein
VL2 is a second antibody light chain variable region, and
CL is a light chain κ or λ constant domain; and c. the third polypeptide has a formula selected from the group consisting of
(VH1-Ca)-X1-(CH2-CH3)-X2-(VH2 -CHl),
(VH2-CHl)-(H-CH2-CH3)-X2-(VHi-Ca), (νΗ!-ΰα)-Χ2-(νΗ2 -¾ΐ)-(Η-¾2"¾3)> and (VH2-CHl)-X2-(VH1-Ca)-X1-(CH2-CH3)'
wherein
VHi is a first antibody heavy chain variable region, and
Ca is a T-Cell Receptor a constant domain, and
Xi is a peptide linker or is absent, and
Cfj2~Cfj3 are heavy chain constant domains 2 and 3, and
H is a hinge region between CHI and CH2, and
X2 is a peptide linker or is absent, and
VH2 is a second antibody heavy chain variable region and
Cm is a heavy chain constant domain 1.
The present disclosure provides a compound comprising an antibody and two Fabs, wherein the VH containing polypeptides of the Fab are linked to the heavy chains of the antibody and wherein the CHI constant regions of the antibody or Fab fragments are replaced with T-cell receptor a constant domains (Ca) and the corresponding light chain constant domains of the antibody or Fabs are replaced with T-cell receptor β constant domains (C ) such that the Ca and C domains allow the corresponding variable regions to associate to form an antigen binding fragment and the CHI and light chain constant regions allow the variable regions associate to form another antigen binding fragment.
The present disclosure provides a compound comprising an antibody and two Fabs, wherein the VH containing polypeptides of the Fab are linked to the heavy chains of the antibody and wherein the CHI regions of the Fab fragments are replaced with T-cell receptor a constant domains (Ca) and the constant domains of the Fab light chains are replaced with T-cell receptor β constant domains (C ) such that the Ca and C domains associate to form part of an antigen binding fragment. The present disclosure also provides a compound comprising an antibody and two Fabs, wherein the VH containing polypeptides of the Fab are linked to the heavy chains of the antibody and wherein the CHI regions of the antibody are replaced with T-cell receptor a constant domains (Ca) and the constant domains of the antibody light chain are replaced with T-cell receptor β constant domains (C ) such that the Ca and C domains associate to form part of an antigen binding fragment. The present disclosure provides a compound comprising three polypeptide chains, in which
a) the first polypeptide chain comprises the formula VHi- Ca wherein
VHi is a first antibody heavy chain variable region, and
Ca is a T-cell receptor a constant domain, and
b) the second polypeptide chain comprises the formula VH2-CHI-H-CH2-CH3 wherein
V¾ is a second antibody heavy chain variable region, and CHI, CH2, and CH3 are heavy chain constant domains 1 , 2, and 3,
respectively, and
H is a hinge region, and
c) the third polypeptide chain comprises the formula VLi-Cp-Xi-VL2-CL,
wherein
VLi is a first antibody light chain variable region,
CP is a T-Cell Receptor β constant domain,
Xi is a linker which can be present or absent,
VL2 is a second antibody light chain region and
CL is either a Ck or light chain antibody constant domain, in which the first and second polypeptide chains associate with the third polypeptide chain to form a binding site (VLi-VHi) for a first binding partner and a binding site for a second binding partner (VL2-VH2).
The present disclosure also provides a compound comprising three polypeptide chains, wherein
a) a first polypeptide chain has the formula VHI-CHI-H-CH2-CH3,
wherein VHi is a first antibody heavy chain variable region, and
CHI, CH2, and CH3 are heavy chain constant domains 1 , 2, and 3,
respectively, and
H is a hinge region, and
b) a second polypeptide chain has the formula VL1-CL-VL2- Cp, wherein
VLi is a first antibody light chain variable region and
CL is either Ck or C^, and
VL2 is a second light chain variable region, and CP is a T-Cell Receptor β constant domain, and
c) the third polypeptide has the formula VH2- Ca, wherein
VH2 is a second antibody heavy chain variable region and Ca is a T-Cell Receptor a constant domain,
in which the first and third polypeptide chains associate with the second polypeptide chain to form a binding site (VL1-VH1) for a first binding partner and a binding site for a second binding partner (VL2-VH2).
The present disclosure also provides a compound comprising three polypeptide chains, wherein
a) a first polypeptide chain has the formula VLi-CL, wherein
VLi is a first antibody light chain variable region and
CL is either Ck or Cx, and
b) a second polypeptide chain has the formula VL2- Cp, wherein
VL2 is a second antibody light chain variable region and
CP is a T-Cell Receptor β constant domain,
c) a third polypeptide chain has the formula VHI-CHI-XI-VH2- Ca, wherein
VHi is a first antibody heavy chain variable region, and
CHI is a heavy chain constant domain 1 , and
Xi is a linker, and
VH2 is a second antibody heavy chain variable region, and
Ca is a T-Cell Receptor a constant domain, and
in which the first and second polypeptide chains associate with the third polypeptide chain to form a binding site (VHi-VLi) for a first binding partner and a binding site for a second binding partner (VH2-VL2).
The present disclosure also provides a compound comprising three polypeptide chains, wherein
a) a first polypeptide chain has the formula VLi- Cp, wherein
VLi is a first antibody light chain variable region, and
CP is a T-Cell Receptor β constant domain,
b) a second polypeptide has the formula VL2-CL, wherein
VL2 is a second antibody light chain variable region, and CL is either Ck or Cx, and c) a third polypeptide has the formula VHi- Ca-Xi-VH2-C , wherein
VHi is a first antibody heavy chain variable region, and
Ca is a T-Cell Receptor a constant domain, and
Xi is a linker, and
VH2 is a second antibody heavy chain variable, and
CHI is heavy chain constant domain 1, and
in which the first and second polypeptide chains associate with the third polypeptide chain to form a binding site (VHi-VLi) for a first binding partner and a binding site for a second binding partner (VH2-VL2).
The present invention also provides a process for producing any of the proceeding compounds comprising cultivating the mammalian cells containing DNA encoding a compound of the present invention under conditions such that the compound is expressed and recovering the compound.
Brief Description of the Figures
Fig. 1. Schematic diagram of Format #1 of a TCR-based IgG-(Fab)2 compound, comprising three polypeptide chains: VI - C , VL2-CL, and (VHI-C(X)-X1-(CH2-CH3)-
X2-(VH2 -CHI), in which VLi is a first antibody light chain variable region, CP is a T- Cell Receptor β constant domain; VL2 is a second antibody light chain variable region,
CL is a light chain κ or λ constant domain; VHi is a first antibody heavy chain variable region, Ca is a T-Cell Receptor a constant domain, XI is a peptide linker or is absent, CH2 -CH3 are heavy chain constant domains 2 and 3, X2 is a peptide linker or is absent, VH2 is a second antibody heavy chain variable region and CHI is a heavy chain constant domain 1.
Fig. 2. Schematic diagram of Format #2 of a TCR-based IgG-(Fab)2 compound, comprising three polypeptide chains: VI - C , VL2-CL, and (VH2-CHI)-(CH2-CH3)- X2-(VH!-Ca). Fig. 3. Schematic diagram of Format #3 of a TCR-based IgG-(Fab)2 compound, comprising three polypeptide chains: VLi - C , VL2-CL, and (VHi-Ca)-
X2_(VH2 -CHI)-(CH2-CH3)- Fig. 4. Schematic diagram of Format #4 of a TCR-based IgG-(Fab)2 compound, comprising three polypeptide chains: VI - Οβ, VL2-CL, and (VH2-CHI)-X2-(VH1-C(X)-
Figure imgf000009_0001
The general structure of an "antibody" is very well-known. For an antibody of the IgG type, there are four amino acid chains (two "heavy" chains and two "light" chains) that are cross-linked via intra- and inter-chain disulfide bonds. When expressed in certain biological systems, antibodies having unmodified human Fc sequences are glycosylated in the Fc region. Antibodies may be glycosylated at other positions as well. The subunit structures and three-dimensional configurations of antibodies are well known. Each heavy chain is comprised of an N-terminal heavy chain variable region ("VH") and a heavy chain constant region ("CH")- The heavy chain constant region is comprised of three domains (CHI, CH2, and CH3) for IgG as well as a hinge region ("hinge") between the CHI and CH2 domains. Each light chain is comprised of a light chain variable region ("VL") and a light chain constant region ("CL"). The CL may be of the kappa ("κ") or lambda ("λ") isotypes.
The variable regions of each light/heavy chain pair to form binding sites. The heavy chain variable region (Vn)and the light chain variable region (VL) of the compounds of the present invention can be subdivided into regions of hypervariability, termed complementarity determining regions ("CDRs"), interspersed with regions that are more conserved, termed framework regions ("FR"). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4. Herein, the 3 CDRs of the heavy chain are referred to as "CDRH1 , CDRH2, and CDRH3" and the 3 CDRs of the light chain are referred to as "CDRL1 , CDRL2 and CDRL3." The CDRs contain most of the residues which form specific interactions with the antigen. The assignment of amino acids to each domain is in accordance with well-known conventions [e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)].
An antibody may be derived from a single copy or clone (monoclonal antibody (mAb)), including e.g., any eukaryotic, prokaryotic, or phage clone. Preferably, an antibody of the present invention exists in a homogeneous or substantially homogeneous population of antibody molecules. A full-length antibody comprises full length or substantially full length constant regions, including the Fc region. An "antigen-binding fragment" of such an antibody is any shortened form of a full length antibody that comprises the antigen-binding variable regions and retains antigen-binding capability. Such shortened forms include, e.g., a Fab fragment, Fab' fragment or F(ab') 2 fragment that includes the CDRs or the variable regions of the antibodies disclosed.
An antibody of the present invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art.
An antibody of the present invention is an engineered antibody that has been engineered to comprise framework, hinge, or constant regions derived from fully human frameworks, hinge, or constant regions containing one or more amino acid substitutions, deletions, or additions therein. Further, an antibody of the present invention is substantially non-immunogenic in humans.
A variety of different human framework sequences may be used singly or in combination as a basis for an antibody of the present invention. Preferably, the framework regions of an antibody of the present invention are of human origin or substantially human (at least 95%, 97% or 99% of human origin.) The sequences of framework regions of human origin may be obtained from The Immunoglobulin
Factsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351.
T-cell receptors are used to recognize foreign antigenic peptides displayed by various antigen presenting cells of the immune system. Binding of T-cells to antigen- presenting cells occurs via interactions between major histocompatability complex
(MHC) moieties of the antigen-presenting cells, which carry an antigenic peptide, and T- cell receptors (TCRs) on the surface of T-cells. Formation of a cell-bridging MHC- peptide-TCR complex typically results in stimulation and activation of the T-cells, a primary step of an adaptive immune response. TCRs are related to immunoglobulins in that they contain hypervariable V-class Ig-fold domains capable of generating an incredible diversity that enables them to recognize diverse peptidic antigens displayed by MHCs. Like antibody Fabs, they also contain C-class Ig-fold constant domains that help stabilize the TCR complex. However, structurally, TCR constant domains have very different primary sequences than immunoglobulin Fab constant domains (-20% identity between the TCR a- and β-domains and IgG CHI and CL constant domains). Additional information regarding T-cell receptors and their constant domains may be found in The T Cell Receptor Factsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001, ISBN 0124413528.
Example 1- Certain Orientations of TCR Constant Regions May Substitute for CH1/CL Regions; Variable Region-Constant Region Linker Length
To determine the ability of the TCR a- and β-constant domains (Cot and C , respectively) to replace the CHI of the heavy chain and light chain constant (CL) domains, varying constructs in a mammalian expression vector for transient expression in HEK293 cells are generated. Both orientations with the Cot or CP domains replacing the CHI domain or the CL domain are constructed. For simplicity, this chimeric antibody format is called IgG-TCR. An anti-human IL-17 IgG4/K (HC of SEQ ID NO:78 and LC of SEQ ID NO:79) antibody is utilized for generating IgG-TCR constructs. The CHI/ CL heterodimeric unit of the antibody is replaced using both Cot/CP and Cp/Cot orientations and varying V-gene/TCR constant domain linker sequences. Each heavy chain ("HC") construct is co-transfected with each light chain ("LC") construct to determine whether any format will allow for assembly, binding, and stability.
The linking regions between IgG V-genes and their constant regions are shorter than the linking regions between TCR V-genes and their respective constant regions. The linker regions between the antibody V-genes and the TCR constant domains, as well as truncated versions of the a- and β-constant connector regions are generated within the IgG-TCR format. The sequences of the constructs with truncations are denoted in Table 1.
All gene constructs are synthesized using PCR-based overlapping oligonucleotide synthesis. Heavy chain genes are subcloned into a vector containing the gene sequence coding for a human IgG4 constant region resulting in the fusion products of the antibody V-genes and the TCR Cot and C genes with the IgG4PAA hinge-Fc domain. Light chains are also subcloned into a vector. The vector contains a common mouse antibody LC signal sequence that is translated in-frame as part of the expressed protein and cleaved prior to secretion. The ligation mixture was used to transform E. coli strain TOP 10 competent cells (Invitrogen Corporation, Carlsbad, CA). Sequences are confirmed by DNA sequencing. Plasmid DNAs are used to transfect 293F cells for transient production of antibody protein.
Table 1: Substitution of Ca and CP for CHi and CL in an anti-human IL-17-IgG4; Orientations and Linker Lengths.
Figure imgf000012_0001
The sequence given by SEQ ID NO:2 represents a truncation of N-terminal Pro-
Asn (PN) from the native human Ca sequence (given by SEQ ID NO: l). The sequence given by SEQ ID NO: 11 represents a truncation of N-terminal Glu-Asp-Leu-Asn (EDLN) from the native human CP sequence (given by SEQ ID NO: 10). The sequences of the variable regions, hinge, CH2, and CH3 are identical for these constructs. The only variation is in the CHi/CL domains.
A vector harboring the LC/TCR DNA sequence and a vector harboring the HC/TCR DNA sequence are transfected (1 :2 plasmid ratio for the HC and LC plasmids) into 293F cells using FreeStyle™ transfection reagents (Life Technologies). Transfected cells are grown at 37 °C in a 5% C02 incubator while shaking at 125 rpm. Secreted protein is harvested by centrifugation at >10,000 rpm for 5 min.
To purify expressed IgG-TCR constructs, supernatants are passed through 2 μιη filters. 1 mL of supernatant is incubated with 100 μL· resuspended, phosphate buffered saline ("PBS")-washed Protein G magnetic beads (Millipore). Beads are washed two times with PBST (phosphate buffered saline/0.02% Tween 80) according to the manufacturer's protocols. Protein is eluted from the beads by adding 130 mL 0.01 M Acetate, pH 3.0. After harvesting, the eluants are immediately neutralized by adding 20 HL O. l M Tris, pH 9.0.
Each expressed IgG-TCR is evaluated by SDS-PAGE. Approximately 5 μg protein is loaded in each well of Novex® 4-20% Tris Glycine gels or 3-8% Tris-Acetate gels according to manufacturer protocols (Life Technologies). For reduced samples, 10% 0.5 M DTT in H20 is added to the same prior to loading.
As shown in Table 2, all the proteins expressed well. However, none of the constructs having in the HC and Ca in the LC (TCRl , TCR3, TCR5, and TCR7) were assembled. Qualitative assembly was determined by SDS-PAGE based on the size of the IgG-TCR protein bands under non-reducing conditions and based on the ability to detect a LC band under reducing conditions (Table 2).
Analytical size exclusion chromatography (SEC) with in-line light scattering (SEC/LS) is performed for each sample. 30-80 μL· of each sample (-0.2-0.8 mg/mL) are injected onto a Sepax Zenix SEC 200 analytical HPLC (7.8x300 mm) column equilibrated in 10 mM phosphate, 150 mM NaCl, 0.02% NaN3, pH 6.8, using an Agilent 1100 HPLC system (Agilent Technologies). Static light scattering data for material eluted from the SEC column are collected using a miniDAWN TREOS static light scattering detector coupled to an Optilab T-rEX in-line refractive index meter (Wyatt Technologies). UV data are analyzed using HPCHEM (Agilent). Molecular weights of the complexes are determined by their static light scattering profiles using ASTRA V (Wyatt Technologies). The SEC results (Table 2) demonstrate that the constructs aggregate to a similar level as a control IgG4 antibody.
The extent of HC/LC assembly is assessed using a kinetic Biacore experiment.
Briefly, kinetic surface Plasmon resonance (SPR) experiments are performed using a Biacore 3000 (GE Healthcare). IgG-TCR proteins are captured onto CM5 sensorchip surfaces with an immobilized goat anti-human IgG-Fc polyclonal antibody (Jackson ImmunoResearch, Cat. #109-005-098). The goat polyclonal antibody immobilization is achieved by injecting the protein (at 50 μg/mL, pH 5) over an NHS/EDC activated sensorchip surface followed by blocking with ethanolamine (as described by
manufacturer). The IgG or IgG-TCRs are captured onto the anti-human Fc sensorchip surface by injecting 10 μL· of protein at 0.1 and 0.5 mg/mL using a 2 μί/ιηί flow rate. Flow rates are increased to 10 μί/ιηίη followed by secondary 30 μL· injections of IL-17 (SEQ ID NO:80) at 10 and 50 nM. Following a 15 minute dissociation period, the flow rate is increased to 60 μΕ/min and sensorchip surfaces are regenerated using two consecutive 10 μΕ injections of 0.1 M glycine, pH 2.0.
The experiment measures the percent activity by relating the amount of captured IgG or IgG-TCR (based on resonance units) with the secondary response generated using a saturating level of IL-17. The percent activity of each IgG-TCR is calculated based on a comparison with the WT IgG:
% Activity
Figure imgf000014_0001
where RUjgQ X R and RUigQ are the resonance units of the captured IgG-TCR or IgG, respectively, and RUJL_I η is the saturating level of resonance units generated by the binding of IL-17 to the pre-loaded anti-IL-17 IgG or IgG-TCR. TCR3, which had the Cot- and CP domains replacing the CL and CHI domains, respectively, demonstrated little activity in the assay indicating a near complete lack of LC/HC assembly (Table 2). TCR2, TCR4, TCR6, and TCR8 (with the a- and β-constant regions replacing the CHI and CL domains, respectively) all had activity. (Table 2). Table 2: Biochemical characterization of TCR1-TCR8.
Figure imgf000015_0001
a Estimated based on characterization of microscale purified material,
b Not measured. Thus, replacing the CHI domain of an immunoglobulin with Cot and replacing the
CL domain of an immunoglobulin with C allows for the native-like heterotetramer formation of an IgG
Example 2- IgG-TCR hinge and LC C-terminus modifications
Antibody I (HC SEQ ID NO. 34; LC SEQ ID NO. 33, trastuzumab), which binds to HER2, is used for the following experiments. The variable regions of Antibody I are appended to the N-termini of TCR4 above. The IgG-TCR construct containing the Antibody I Fv is denoted as tTCR-G4(+) and has HC and LC sequences of SEQ ID NO:36 and SEQ ID NO:35, respectively. Next, the IgG4 hinge (SEQ ID NO:5) of tTCR- G4 is replaced with an IgGl hinge (SEQ ID NO: 6) resulting in tTCR-Gl(+) which has a HC of SEQ ID NO:37 and a LC of SEQ ID NO:35.
To determine whether changes to C in the LC can improve assembly, truncations are made in the C-terminus of ΰβ. A new construct, with four residues truncated from the C-terminal sequence of C is created (SEQ ID NO: 12). A construct tTCR-Gl(-) contains the same tTCR-Gl HC (SEQ ID NO: 37) with the modified tTCR-Gl LC (SEQ ID NO:38) truncated at its C-terminus. Similarly, pTCR-Gl(-) containing the Antibody II (HC SEQ ID NO:42; LC SEQ ID NO:41) variable regions (HCVR SEQ ID NO:40; LCVR SEQ ID NO:39) is constructed.
All gene constructs are synthesized using protocols similar to those described in Example 1. Protein expression and purification are performed as described in Example 1. Protein characterization is performed as described in Example 2, except that the kinetic Biacore assay used to measure IL-17 activity is replaced with a solution equilibrium assay that measures HER2 equilibrium affinity and stoichiometry. Briefly, Antibody I is directly immobilized to 12000 RUs onto an NHS/EDC activated CM5 sensor chip surface by injection of a 50 μg/mL solution of Antibody I in 10 mM Acetate, pH 5.0. A linear RU response is observed by injecting human (h)HER-2-Fc protein (Cat. #1129-ER-050, R&D systems) at 2 μΕ/min at multiple concentrations between 1 and 100 nM over the sensorchip surface and monitoring the response after 80 seconds of binding. To evaluate the activity/assembly of the IgG-TCR, 20 nM hHER-2-Fc are mixed with varying concentrations (ranging from 1 nM to 100 nM) of each test article. The Antibody I sensorchip surface measures the concentration of unbound or free hHER-2-Fc ([R]F) in solutions containing 20 nM hHER-2-Fc and IgG-TCR. Unbound hHER-2-Fc is equal to the total amount of receptor in solution ([R]T) minus the bound concentration ([R]B). The equilibrium dissociation constant, KD, and binding stoichiometry, n (used to determine the assembly), between the IgG-TCRs and hHER-2-Fc are determined using the linear relationship between [R]p and linear slope of RU/time (known as the velocity or Vi) within the first 80 seconds of the experiment:
Figure imgf000016_0001
T where m = slope of the hHER-2-Fc concentration-dependent standard curve and
[IgG_TCR]T = total IgG_TCR concentration. Table 3:-tTCR-Gl(+), tTCR-Gl(-), and pTCR-Gl(-) construct compositions.
Figure imgf000017_0001
ii The sequence for CH2-CH3 was that of SEQ ID NO: 15, except that the terminal Lys (K) was deleted.
Table 4: Assembly of IgG-TCR constructs
Figure imgf000017_0002
"n.d " denotes not determined. Truncation of the C-terminal four amino acids of C results in a 5-fold average increase in protein expression as determined according to the procedure in Example 2 (data not shown). The proteins with the LC-C truncation are more uniformly the expected molecular weight based on in-line light scattering measurements and do not appear to be incompletely assembled. Additionally, truncating four LC-C -terminal amino acids eliminates apparent proteolysis, mis-assembly, or protein degradation that had been observed as multiple absorbance peaks eluting for non-truncated molecules (data not shown).
A Biacore-based equilibrium binding experiment between tTCR-Gl(+), tTCR- Gl(-), and pTCR-Gl(-) proteins with the hHER-2-Fc protein demonstrates that both the tTCR-Gl(-) and pTCR-Gl(-) proteins are fully assembled, while the tTCR-Gl(+) protein is only -75% assembled (Table 4). As a control, Antibody I and Antibody II are shown to block hHER-2-Fc binding to surfaces immobilized with the antibodies, but to not block each other with a stoichiometry matching the hHER-2-Fc concentration used in the assay (20 nM) (Table 4). The tTCR-Gl compounds could not inhibit hHER-2-Fc from binding an Antibody II-labeled surface and the pTCR-Gl(-) compound could not inhibit hHER-2- Fc binding to an Antibody I-labeled surface, indicating that the specificity for their particular epitopes is intact. Different batches of tTCR-Gl (+) appeared to give varying levels of assembly, as measured by the stoichiometry of hHER2-Fc blocking in the assay, perhaps because of differences in plasmid levels that were transfected (data not shown). The tTCR-Gl(-) and pTCR-Gl(-) proteins consistently block binding of 20 nM hHER2- Fc to surfaces with Antibody I and Antibody II, respectively, at 20 nM concentrations indicating they are 100% assembled. Overall, the presence of an IgGl hinge (including the interchain cysteine that forms a disulfide with LC) and the LC-C truncation results in fully assembled IgG-TCRs.
Example 3- Specificity of LC and HC Assembly
To determine the extent to which introduction of TCR-constant domains into a Fab enables specific HC/LC assembly that discriminates from wild-type HC/LC assembly (i.e., HCs and LCs containing CHI and CL domains, respectively), tTCR-Gl(-) and pTCR-Gl(-) HC plasmids are transfected with both their cognate C constant domain containing LCs and the natural (non-TCR-domain containing) LC plasmids (Table 5). Additionally, the wild-type IgGl Antibody I (trastuzumab) and Antibody II (pertuzumab) HC plasmids are transfected with their cognate natural LCs and their LCs containing C constant domains (Table 5).
An OctetRed (ForteBio) biosensor assay is used to evaluate the specificity of the optimized Antibody I and Antibody II IgG-TCR constructs within this Example. The method includes the use of an anti-human IgG-Fc specific biosensor (ForteBio) to capture the IgG-TCR HC or wild-type IgG HC transfected in the presence of both the wild-type and TCR -containing LCs (Table 5) at 10 μg/mL in Octet buffer for 5 minutes. The captured IgGs or IgG-TCRs are then used to capture an anti-kappa CL domain- specific murine mAb (Sigma-Aldrich Cat. #SAB4700607) or an anti-CP constant domain-specific murine mAb (ThermoScientific Cat. #TCR1151) at 10 μg/mL in Octet buffer for 5 minutes. The % non-specific binding is assessed as the saturated signal of the anti-CP or anti-CK antibodies compared to the controls.
Table 5: Co-transfection of Antibody I and Antibody II IgG-TCR and IgG HCs with both natural LCs or LCs containing TCR-β constant domains.
Figure imgf000019_0001
Wild-type IgG HCs show strong selective binding to their cognate wild-type LC when expressed in the presence of a CP-containing LC with the same variable domains. Additionally, Coc-containing IgG HCs show selective binding to their cognate C - containing LCs when expressed in the presence of a wild-type LC. Example 4- Construction of IgG-(Fab)2 compounds
To determine whether Coc and C domains can be used to generate compounds in which a Fab with one specificity is linked to an antibody with another specificity (IgG- (Fab)2) and whether the HCs and LCs of such an IgG-(Fab)2 can assemble appropriately to maintain their binding activities, IgG-(Fab)2 with different Coc and C configurations are constructed. In one configuration, the HC portion of the Fab is linked to the N- terminus of the HC of the antibody and in another configuration, the HC portion of the Fab is linked to the C-terminus of the antibody. For each configuration, the CHI region of the antibody or Fab is replaced with a Coc domain, while the corresponding LC constant domain is replaced with C . Each of the compounds contains a HC containing both a VH/ CHI domain and a VH/COC domain and two LCs, one containing a VL/CL pair and the other containing a Vi/C -constant domain pair. The combinations of HCs and LCs that compose each compound are listed in Table 7 and 8 and illustrated in Figures 1-4. All constructs fall into one of four formats, as shown below:
Table 6: IgG-(Fab)2 compositions
Figure imgf000020_0002
All the methods for generation of the IgG-Fab constructs including, gene synthesis, subcloning, and plasmid preparations are described in Example 1. Table 7: TCR-based IgG-(Fab)2 Constructs
Figure imgf000020_0001
tVH-Ca-X2- pVH-Cm-(H- Cm-Cm) c
32 - 2 -13 - 40 - 14 - 6- 15
N-tTCRpGl 3 tVL-CB DVL-K
31 - 12 39 -21
tVH-Ca-X2- PVH-CHI- (H - Cm-Cm) c
32 -2 -81 - 40- 14 - 6- 15
N-tTCRpGlds 3 tVL-CB DVL-K
31 - 12 39 -21
pVH-CHl-X2-tVH-Ca -Xl-(Cm-Cm) c
- 6 - 15
N-pGltTCR 4
Figure imgf000021_0001
pVH-Cm-X2-tVH-Ca -XI -(Cm-Cm) c
- 6 - 15
N-pGltTCRds 4
Figure imgf000021_0002
pVH-Ca -Xl-iCm-Cm)-X2-tVH-Cm-a
40 -2 - 6- 15 -13 - 32 - 14
C-pTCRtGl 1 pVL-CB tVL- K
39 - 12 31 - 21
tVH-Cm - fH-Cm-Cm)-X2-pVH-Ca b
32 - 14 -6 - 15 - 13- 40- 2
C-tGlpTCR 2 tVL- K pVL-CB
31 -21 39-12
pVH-Ca-X2- tVH-Cm-(H- Cm-Cm) c
40- 2 -13 - 32- 14 - 6- 15
N-pTCRtGl 3 pVL-CB tVL-K
39- 12 31 -21
pVH-Ca-X2- tVH-Cm-(H- Cm-Cm) c
40-2 -81- 32-14 - 6- 15
N-pTCRtGlds 3 pVL-CB tVL-K
39- 12 31 -21
tVH-Cm-X2-pVH-Ca -XI -(Cm-Cm) c
32 - 14 -13 -40 - 2 - 6 - 15
N-tGlpTCR 4 tVL- K pVL-CB
31 -21 39-12
tVH-CH,-X2-pVH-Ca -XI -f Cm-Cm) c
32 - 14 -82-40 -2 - 6 - 15
N-tGlpTCRds 4 tVL- K pVL-CB
31 -21 39-12 a C-terminal Glu-Pro-Lys-Ser-Cys-Asp-Gly-Gly-Gly (EPKSCDGGG) on HC. b C-terminal Glu-Ser-Ser-Cys-Asp-Val-Gly-Gly-Gly (ESSCDVGGG) on HC. c C-terminal is des-Ly (des K).
Table 8: Sequence composition of IgG-(Fab)2 compounds
Figure imgf000022_0001
The following nomenclature is used when referring the IgG-Fab compounds of the present invention. "C-" or "N-" denotes an additional Fab region (either wild-type Fab or Fab containing TCR-constant domains) appended to the C- or N-terminus of the HC, respectively, "t" refers to Antibody I Fv (trastuzumab Fv), "p" refers to Antibody II (pertuzumab Fv), "Gl" denotes a wild-type Fab while "TCR" denotes a Fab containing TCR-constant domains. The order with which the Gl and TCR sequences are listed within each name indicates the order within the primary sequence that the Fab regions occur. For example, C-tTCRpGl indicates that a Fab of Antibody II (pertuzumab Fab), which has antibody CHI/CL domains, is linked to the C-terminal end of the HC of Antibody I (trastuzumab) whose CHI/CL domains are replaced by TCR Coc/C , respectively.
IgG-(Fab)2 compound characterization, including protein expression, protein purification, and in vitro biochemical are performed as described in Example 1. The binding/assembly properties of the IgG-Fab compounds are determined using the SPR- based solution equilibrium methodology described in Example 2.
Table 9: Characterization of the IgG-Fab recognizing two epitopes of HER2
Figure imgf000023_0001
determined by SEC with in-line static light scattering. mAb data +5 kDa; BsAb data +10 kDa.
bDetermined by equilibrium solution SPR experiments.
°Protein displayed varying HC/LC compositions based on SEC.
dProtein displayed varying HC/LC compositions based on SEC.
" "Indicates WT-IgG Fab binding stoichiometry/assembly results
"n.d " denotes not determined.
The IgG-(Fab)2 compounds could all block hHER-2-Fc from binding both surfaces (Table 9). The data suggest that four binding sites are intact. Example 5- Activity of IgG-(Fab)2 Compounds in cell assays
Next, we assayed IgG-(Fab)2 compounds for their ability to inhibit HER-2-driven tumor cell growth. The NCI-N87 (gastric) and BT474 (breast) tumor cell lines both highly over express HER-2 on their cell surfaces and have been shown to be sensitive to treatment using the trastuzumab/pertuzumab combination.
For the inhibition assays, HER-2-positive NCI-N87 (ATCC Cat. #CRL-5822) or
BT474 (ATCC Cat. #HTB-20) tumor cell lines are cultured according to the guidelines provided by the ATCC. For flow cytometry, cells (-75% confluent) are lifted from their culture flasks using cell dissociation buffer (Cat. #13151014 Life Technologies), counted, and plated in 96- well round bottom tissue culture plates at 0.5 x 106 cells per well.
Mouse Ig Gl-PE (BD), Mouse anti-EGFR-PE (BD), Mouse anti-Her-2/neu-PE (BD), and Mouse anti-Her-3/erbB3-PE (BD) all are used at 20 μ1/0.5χ106 cells. All mAbs are diluted in flow cytometry buffer (Dulbecco PBS w/2% FBS & 0.05% sodium azide & 10% NGS) and incubated for 45 minutes. The cells are centrifuged at 1500 rpm for 5 minutes at 4 C and washed three times with flow cytometry buffer. After final wash the cells are resuspended in flow cytometry buffer containing propidium iodide (PI,
Molecular Probes) to stain for dead cells. Samples are run on the LSR Fortessa acquiring with Diva software (both from Becton Dickinson) and analyzed with FloJo (version 7.6.3).
For fetal bovine serum (FBS)-mediated tumor cell proliferation experiments, NCI- N87 or BT-474 cell lines are seeded on 96-well plates at 1 x 103 cells per well and precultured in RPMI-1640 medium containing 10% FBS overnight. To evaluate the impact of the IgG-(Fab)2 compounds on cell proliferation, 100, 10, 1, and 0.1 nM solutions of each test compound (or combinations of 100, 10, 1, and 0.1 nM of each test compound) in RPMI-1640 medium containing 10% FBS are added to the cells.
Alternately, 300, 30, 3, and 0.1 nM solutions of each test compound or combinations of test compounds are evaluated in the same buffer solutions. After 5 days of treatment, cell viability is determined with a Cell Titer Glo reagent (Promega). The percentage of growth inhibition is calculated according to the formula [1 -(signal with mAb (or IgG-(Fab)2)) /(signal with FBS only)]* 100.
To measure the levels of phosphorylated EGFR, HER-2, and HER-3 on the BT- 474 cell line the following protocol is used. BT-474(breast cancer) cell lines are seeded in 12- well culture plates at 2.5 x 105 cells per well and grown in RPMI-1640 medium containing 10% FBS overnight. The next day, cells are treated with 100 nM mAbs or IgG-(Fab)2 for 24, 48 and 72 hours at 37°C in 10% FBS containing medium. Cell lysates are made using cell lysis buffer (MSD Cat. #R60TX-3), and protein concentrations are measured using BCA protein assay (Pierce). Phospho-HER-2, Phospho-HER-3 and Phospho-EGFR in cell lysates are measured using a Phospho-HER-2, Phospho-HER-3 and Phospho-EGFR multiplex MSD kit (Meso Scale Discovery). Plates are loaded with 16 μg total protein in duplicate, and incubated for 2 hours at room temperature with shaking. Next, plates are washed and detection antibody is added and incubated for 2 hours at room temperature with shaking. Plates are read on a Sector Imager6000 (Meso Scale Discovery).
Table 10: Inhibition of BT474 breast and NCI-N87 gastric cancer cell lines by the IgG-(Fab)2 compounds.
"n.d " denotes not determined.
As shown in Table 10: Antibody I (trastuzumab) and Antibody II (pertuzumab) individually inhibited FBS-mediated tumor cell growth of both the BT474 and NCI-N87 cell lines. In addition, the combination of Antibody I and II resulted in an increase in anti-proliferative activity. The individual IgG-TCR proteins, tTCR-Gl(-) and pTCR- Gl(-), and the combination of these proteins demonstrated significant decreases in antiproliferative activity presumably due to the different Fab/hinge dynamics introduced using the subtly different connecting regions within the IgG-TCR format. Table 10 also shows that the various IgG-(Fab)2 compounds possess a spectrum of activities on HER-2- mediated tumor cell growth ranging from antagonistic to highly-antagonistic with the N- pTCRtGl being even more inhibitory in the NCI-N87 cell line than the Antibody I and II combination. Example 6- IgG-(Fab)2 combining the specificities of the anti-HER-2 mAb pertuzumab and the anti-EGFR mAbs cetuximab and matuzumab.
To determine whether the IgG-(Fab)2 of the present invention are able to bind to two different targets, IgG-(Fab)2 compounds targeting EFGR and HER-2 are generated. Different constructs containing a cetuximab Fab and wild type pertuzumab (with IgGl CHI domain and kappa CL domain) as well as two additional constructs containing a matuzumab Fab and wild-type pertuzumab (with IgGl CHI domain and kappa CL domain), each in the IgG-TCR format are generated. Cetuximab is a chimeric mouse/human mAb directed against EGFR (HC of SEQ ID NO: 60 and LC of SEQ ID NO:59), while matuzumab is a humanized anti-EGFR mAb (HC of SEQ ID NO:69 and LC of SEQ ID NO:68). The HCs and LCs that comprise the IgG-Fab are provided in Table 11 and 12 as are the sequences of the antibody controls and an IgG-TCR control, mTCR-Gl(-). "c" refers to Antibody III (cetuximab Fv), and "m" refers to Antibody IV (matuzumab Fv) containing Fab. The remainder of the nomenclature is consistent with Example 4. Table 11: Composition of the anti-HER-2/anti-EGFR IgG-Fabs and relevant controls
Figure imgf000027_0001
Table 12: HC and LC sequence composition of the anti-HER-2/anti-EGFR IgG- (Fab)2 and controls
Figure imgf000027_0002
The construction, expression, purification, and biophysical characterization of the anti-HER-2/anti-EGFR IgG-(Fab)2 compounds is performed as described in the Example 4. The four IgG-(Fab)2 compounds designed to recognize both EGFR and HER-2 are expressed and purified (by protein G magnetic beads only - no secondary purification steps) at the 2 mL and 10 mL scales.
The proteins are characterized by SDS-PAGE and analytical SEC as in Example
1.
To determine whether the IgG-(Fab)2 compounds are able to engage both targets simultaneously, an ELISA assay is run. Specifically, clear 96-well round bottom high binding microtiter plates (Greiner) are coated overnight at 2-8 °C with 50 μΕ/well 1 μg/mL hEGFR-Fc (Cat. #344-ER-050, R&D systems) in a 50 mM Na2C03 pH 9.4 buffer. The plate is washed 4X times with PBST and blocked with 100 μΕ/well casein buffer (Pierce) for 1 hr at 37 °C. The plate is then washed 4X times with PBST and test compounds are added at 50 μΕ/well and 30 μg/mL (20 nM) and serially diluted 1 :2 down the plate. The test compounds are incubated on the plate for 1 hr at 37 °C. The plate is washed 4X times with PBST and 50 μΙ7 well 0.2 μg/mL hHER-2-Histag (Sino Biologies) is added for 1 hr at 37 °C. The plate is then washed 4X times with PBST followed by the addition of a 50
Figure imgf000028_0001
secondary anti-Histag-HRP antibody (PENTA-His-HRP, Qiagen) diluted 1 :1000 in PBST. The secondary antibody is incubated for 1 hr at 37 °C. The plate is then washed 4X times with PBST and 100 μίΛνβΙΙ 1-component 3,3',5,5'- Tetramethylbenzidine (TMB) substrate is added (KPL laboratories). After approximately 10 minutes, 100 μΕ/well 1% H3PO4 (in H20) is added to quench the reaction. Absorbance (450 nm) of every well in the plate is read using a SpectraMax UV plate reader
(Molecular Devices).
Table 13: Characterization of TCR-based IgG-Fabs that recognize EGFR and HER-2.
Figure imgf000028_0002
N-mTCRpGl 90/10 254+20 2.0+2.0
cetuximab IgGl 99.5/0.5 Not determined No binding observed mTCR-Gl(-) Not determined Not determined No binding observed
As shown in Table 13, the IgG-(Fab)2 compounds have the correct molecular weight based on SDS-PAGE and analytical SEC and are primarily monodisperse with ~3- 10% soluble aggregates. Also, all four IgG-(Fab)2 compounds are capable of binding both EGFR and HER-2 based on positive signals in the sandwich ELISA and the measured potencies are listed in Table 13.
The results of the expression and biophysical characterization indicate the IgG- (Fab)2 compounds not only express at their expected molecular weights, but display relatively ideal starting biophysical properties. The dual- specificity binding ELISA demonstrates the ability to bind both antigens for these IgG-Fab constructs.
SEQ ID NO:l TCRoc
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP
SEQ ID NO:2 TCR a N-term,
IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP SEQ ID NO: 3 TCR a N-terminal truncated IgG4 Fc region
IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVK
SEQ ID NO:4 TCRa N term, C-term truncation + IgGl hinge seq
IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSC
SEQ ID NO: 5 IgG4 hinge fragment
ESSCDVK
SEQ ID NO:6 IgGl hinge fragment
EPKSC
SEQ ID NO:7 N-terminal truncated -(K) TCRa
IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDV
SEQ ID NO: 8 N-terminal truncated -(K)TCRoc + GGG
IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVGGG
SEQ ID NO: 9 N-terminal truncated TCRa + Linker
IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPGGGGSGGGGSGGGGSGGGG SGGGGS SEQ ID NO:10 TCR
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHS GVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDR AKP VTQI VS AE AWGR ADC GFTS
SEQ ID NO:l 1 N-term truncated TCR
KVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQD RAKP VTQI VS AEAWGR ADC GFTS
SEQ ID NO: 12 N-term, C-term truncation TCR β
KVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVST
DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQD
RAKPVTQIVSAEAWGRADC
SEQ ID NO: 13 Linker
GGGGSGGGGSGGGGSGGGGS
SEQ ID NO: 14 CHI
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
SEQ ID NO: 15 CH2-CH3
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
SEQ ID NO: 16 CH1-CH2-CH3
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK SEQ ID NO:17 (CHl-L)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGGSGGGGSG GGGSGGGGSGGGGS
SEQ ID NO: 18 (CH2-CH3-L)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGKGGGGSGGGGSGGGGSGGGGS
SEQ ID NO: 19 (CH1CH2CH3-L)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GKGGGGSGGGGSGGGGSGGGGS
SEQ ID NO:20 (A-X1-CH2CH3-X2)
IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM
DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS
GGGGSGGGGSGGGGS
SEQ ID NO:21 CK region
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:22 region
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID NO:23: anti-IL-17 LC Vi/Cp
DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKV SNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHS GVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDR AKP VTQI VS AE AWGR ADC GFTS
SEQ ID NO:24: anti-IL-17 LC Vi/Cp_truncated
DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKV SNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK KVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQD RAKP VTQI VS AEAWGR ADC GFTS
SEQ ID NO:25: anti-IL-17 LC Vi/Ccc
DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKV SNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKP NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVK
SEQ ID NO:26: anti-IL-17 Vi/Ccc_truncated
DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKV SNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKI QNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMD FKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVK
SEQ ID NO:27: anti-IL-17 HC VH/Cp/IgG4Fc
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINP MYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGV YWGQGTLVTVSSEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVEL SWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRC QVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESKYGPPCPPCPAPE
AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG
QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ ID NO:28 anti-IL-17 HC VH/Cp truncated/IgG4Fc
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINP
MYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGV
YWGQGTLVTVSSKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWW
VNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF
YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESKYGPPCPPCPAPEAAGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP
REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ ID NO:29 anti-IL-17 HC VH/Coc/IgG4Fc
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINP
MYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGV
YWGQGTLVTVSSPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD
VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCD
VKESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE
VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN
HYTQKSLSLSLG
SEQ ID NO:30 anti-IL-17 HC VH/Ca_truncated/IgG4Fc
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINP MYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGV YWGQGTL VT VS S IQNPDP A V YQLRDS KS SD KS VCLFTDFDS QTN VS QS KD SD VYI TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKE SKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLG
SEQ ID NO:31 trastuzumab LCVR
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
SEQ ID NO:32 trastuzumab HCVR
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSS
SEQ ID NO:33 Trastuzumab LC
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:34 trastuzumab HC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNS GALTS G VHTFP A VLQS S GLYS LS SWT VPS S SLGTQT YICNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK
SEQ ID NO:35 anti-HER-2 trastuzumab LC VL/CP
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKKVFPP EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP VTQIVSAEAWGRADCGFTS
SEQ ID NO:36 anti-HER-2 trastuzumab HC VH/Coc/IgG4Fc
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD
VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCD
VKESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE
VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN
HYTQKSLSLSLG
SEQ ID NO:37 anti-HER-2 trastuzumab HC VH/Coc/IgGlhinge+Fc
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD
VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPG
SEQ ID NO:38 anti-HER-2 trastuzumab LC Vi/Cp with C-terminal truncation
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKKVFPP
EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL
KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADC SEQ ID NO:39 pertuzumab LCVR
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRY TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK
SEQ ID NO:40 pertuzumab HCVR
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTLVTVSS
SEQ ID NO:41 pertuzumab LC
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRY TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:42 pertuzumab HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK
SEQ ID NO:43 IgG_TCR BsAb HC denoted C-tTCRpGl (C-terminal Fab fusion is wild- type pertuzumab Fab)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD
VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAA
SGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKN
TLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLA
PS S KSTS GGT A ALGCL VKD YFPEP VT VS WNS GALTSG VHTFP A VLQS S GL YSLS S
VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGG
SEQ ID NO:44 IgG_TCR BsAb HC denoted C-pGltTCR (C-terminal Fab fusion is IgG_TCR trastuzumab Fab)
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL
RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA
DTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSIQNP
DPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS
NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVGGG
SEQ ID NO:45 anti-HER-2 pertuzumab LC Vi/Cp with C-terminal truncation
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRY
TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKKVFPP
EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL
KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADC
SEQ ID NO:46 anti-HER-2 pertuzumab HC VH/Coc/IgGlhinge+Fc
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWGQGTL VT VS S IQNPDP A V YQLRDS KS SD KS VCLFTDFDS QTN VS QS KD SD VYI TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPG
SEQ ID NO:47 C-tTCRpGl HC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD
VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAA
SGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKN
TLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLA
PS S KSTS GGT A ALGCL VKD YFPEP VT VS WNS GALTSG VHTFP A VLQS S GL YSLS S
VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGG
SEQ ID NO:48 C-pGltTCR HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL
RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA
DTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSIQNP
DPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS
NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVGGG SEQ ID NO:49 N-tTCRpGl HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTL VT VS S IQNPDP A V YQLRDS KS SD KS VCLFTDFDS QTN VS QS KD SD VYI
TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPGGGGSGG
GGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW
VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED
TAVYYCSRWGGDGFY AMD YWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:50 N-pGltTCR HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNI
KDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ
MNSLRAEDTAVYYCSRWGGDGFY AMD YWGQGTL VTVSSIQNPDPAVYQLRDS
KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKS
DFACANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:51 C-pTCRtGl HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTL VTVS S IQNPDP A V YQLRDS KS SD KS VCLFTDFDS QTN VS QS KD SD VYI
TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFN
IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ
MNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGG
SEQ ID NO:52 C-tGlpTCR HC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNS GALTS G VHTFP A VLQS S GLYS LS SWT VPS S SLGTQT YICNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGG
SLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFT
LSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSIQNP
DPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS
NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVGGG
SEQ ID NO:53 N-pTCRtGl HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTL VT VS S IQNPDP A V YQLRDS KS SD KS VCLFTDFDS QTN VS QS KD SD VYI
TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPGGGGSGG
GGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW
VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED
TAVYYCSRWGGDGFY AMD YWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:54 N-tGlpTCR HC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNS GALTS G VHTFP A VLQS S GLYS LS SWT VPS S SLGTQT YICNVNHKPSNTKVD
KKVGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAAS
GFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNT
LYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSIQNPDPAVYQLR
DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN
KSDFACANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:55 Trastuzumab VL/TCR-P LC
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKKVFPP
EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL
KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADC
SEQ ID NO:56 Pertuzumab VL/TCR-P LC
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRY
TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKKVFPP
EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL
KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADC
SEQ ID NO:57 cetuximab LCVR
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIP SRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK SEQ ID NO:58 cetuximab HCVR
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSG
GNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY
WGQGTLVTVSA
SEQ ID NO:59 Cetuximab LC
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIP SRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 60 Cetuximab HC
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSG
GNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY
WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG
SEQ ID NO:61 C-cTCRpGl HC
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSG
GNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY
WGQGTLVTVSAIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT
DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFT
FTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYL
QMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSK STS GGTA ALGCLVKD YFPEP VT VSWNS G ALTS GVHTFPA VLQS S GLYSLS S V VT V PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGG
SEQ ID NO: 62 Cetuximab VL/TCR- LC
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIP
SRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKKVFPPEV
AVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKE
QPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT
QIVSAEAWGRADC
SEQ ID NO: 63 C-mTCRpGl HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFN
PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG
RYFDYWGQGTLVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKD
SDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA
ASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSK
NTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGG
SEQ ID NO: 64 matuzumab VL/TCR- LC
DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLAS
GVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKKVFPPE
VAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLK
EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT
QIVSAEAWGRADC SEQ ID NO: 65 N-cTCRpGlds HC
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSG
GNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY
WGQGTLVTVSAIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT
DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDGGGG
SGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYT
MDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSL
RAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
A ALGCL VKD YFPEP VT VSWNS G ALTS G VHTFP A VLQS S GL YS LS S V VTVPS S SLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO: 66 matuzumab LCVR
DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLAS G VPSRFS GS GS GTD YTFTIS SLQPEDI ATY YCQQWS SHIFTFGQGTKVEIK
SEQ ID NO: 67 matuzumab HCVR
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFD YWGQGTL VT VS S
SEQ ID NO: 68 matuzumab LC
DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLAS GVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:69 matuzumab HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPG
SEQ ID NO:70 N-mTCRpGlds HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFN
PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG
RYFDYWGQGTLVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKD
SDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC
DGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGF
TFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLY
LQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:71 N-pTCRtGlds HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTL VT VS S IQNPDP A V YQLRDS KS SD KS VCLFTDFDS QTN VS QS KD SD VYI
TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDGGG
GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT
YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSL
RAEDTAVYYCSRWGGDGFY AMD YWGQGTL VTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:72 N-tGlpTCRds HC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNS GALTS G VHTFP A VLQS S GLYS LS SWT VPS S SLGTQT YICNVNHKPSNTKVD
KKVEPKSCDGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR
LS C A AS GFTFTD YTMD WVRQAPGKGLEW V AD VNPNS GGS IYNQRFKGRFTLS V
DRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSIQNPDPA
VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSA
VAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:73 N-tTCRpGlds HC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD
VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCD
GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFT
FTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYL
QMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSK
STS GGTA ALGCLVKD YFPEP VT VS WNS GALTS GVHTFPA VLQS S GLYSLS S V VT V
PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRV VS VLT VLHQD WLNGKE YKC KVS NKALP APIEKTIS KAKGQPREPQ V YTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:74 N-pGltTCRds HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSC
AASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSK NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSIQNPDPAV
YQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV
AWSNKSDFACANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRV VS VLT VLHQD WLNGKE YKC KVS NKALP APIEKTIS KAKGQPREPQ V YTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:75 N-pTCRtGlds HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNP
NSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD
YWGQGTL VT VS S IQNPDP A V YQLRDS KS SD KS VCLFTDFDS QTN VS QS KD SD VYI
TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDGGG
GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT
YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSL
RAEDTAVYYCSRWGGDGFY AMD YWGQGTL VTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTIS KAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:76 N-tGlpTCRds HC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT
NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNS GALTS G VHTFP A VLQS S GLYS LS SWT VPS S SLGTQT YICNVNHKPSNTKVD
KKVEPKSCDGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR
LS C A AS GFTFTD YTMD WVRQAPGKGLEW V AD VNPNS GGS IYNQRFKGRFTLS V
DRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWGQGTL VTVSSIQNPDPA
VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSA
VAWSNKSDFACANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:77 mTCR_Gl(-) HC (IgG-TCR)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFN
PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG
RYFDYWGQGTLVTVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKD
SDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPG
SEQ ID NO:78 IL-17 IgG4 antibody HC
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINP
MYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGV
YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLG
SEQ ID NO:79 IL-17 kappa LC
DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKV SNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 80 ML- 17 with C-terminal FlagHis tag
GITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHR NEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSF RLEKILVSVGCTCVTPIVHHVAAFIDYKDDDDKHVHHHHHH SEQ ID NO:81 Hinge-Linker
ESSCDGGGGSGGGGSGGGGSGGGGS
SEQ ID NO:82 Hinge-Linker
EPKSCDGGGGSGGGGSGGGGSGGGGS

Claims

We claim:
1. A compound comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, in which
a. the first polypeptide chain has the formula VLi - Οβ, wherein i. VLi is a first antibody light chain variable region, and ii. is a T-Cell Receptor β constant domain (Οβ); and b. the second polypeptide has the formula Vl ?- CL, wherein i. VL2 is a second antibody light chain variable region, and ii. CL is a light chain κ or λ constant domain; and c. the third polypeptide chain has a formula selected from the group
consisting of
(VH1-Ca)-Xl-(CH2-CH3)-X2-(VH2 -Cm),
(VH2-CHi)- (H-CH2-CH3)-X2-(VHi-Ca), (VH!-Ca)-X2-(VH2 -CHIMH-CHI-CHS), and
(VH2-CHi)-X2-(VH1-Ca)-Xl-(CH2-CH3), wherein
VHj is a first antibody heavy chain variable region,
Ca is a T-Cell Receptor a constant domain (Coc),
Xi is a peptide linker or is absent,
Cr rlT„-Cr rlT,J are heavy ^ chain constant domains 2 and 3,
H is a hinge region,
X2 is a peptide linker or is absent,
VH2 is a second antibody heavy chain variable region and CH1 is heavy chain constant domain 1; and in which the first and second polypeptide chains independently associate with the third polypeptide chain to form a first antigen binding site (VH1-VL1) and a second antigen binding site (VH2-VL2).
2. The compound of claim 1, wherein Ca is selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:7.
3. The compound of claim 1 or 2, wherein is selected from the group consisting of SEQ ID NO:10, SEQ ID NO: l l, and SEQ ID NO:12.
4. The compound of any one of claims 1-3, wherein Ca is SEQ ID NO:2.
5. The compound of any one of claims 1-4, wherein is SEQ ID NO:l 1.
6. The compound of any one of claims 1-4, wherein is SEQ ID NO:12.
7. The compound of any one of claims 1-6, wherein Cfj2~Cfj3 is SEQ ID NO: 15, CHI is SEQ ID NO: 14, X2 is SEQ ID NO:13, and Xi is SEQ ID NO:5 or SEQ ID NO:6.
8. The compound of any one of claims 1-7, in which Xi is SEQ ID NO:6.
9. The compound of any one of claims 1-8, wherein CL is SEQ ID NO:21.
10. The compound of any one of claims 1-9, comprising two of each of the first, second, third polypeptides, in which a first polypeptide chain and a second polypeptide chain independently associate with one of the third polypeptide chains to form a first antigen binding site and a second antigen binding site, and the other first polypeptide chain and the other second polypeptide chain associate with the other third polypeptide chain to form another first antigen binding site and another second antigen binding site.
11. A DNA molecule comprising a polynucleotide sequence encoding a polypeptide chain having a formula selected from the group consisting of
(VH1-Ca)-X2-(VH2 -CHl)-(CH2-CH3)' (VH1-Ca)-X1-(CH2-CH3)-X2-(VH2 -CHl),
(VH2-CHl)-(CH2-CH3)-X2-(VHi-Ca), and
(VH2-CHl)-X2-(VH1-Ca)-X1-(CH2-CH3)'
which
YH\ is a first antibody heavy chain variable region,
Ca is a T-Cell Receptor a constant domain (Coc),
Xi is a peptide linker or is absent,
Cfj2~Cfj3 are heavy chain constant domains 2 and 3,
X2 is a peptide linker or is absent,
VH2 is a second antibody heavy chain variable region
Cm is a heavy chain constant domain 1.
12. A mammalian cell comprising DNA encoding three polypeptide chains, a. wherein a first polypeptide chain has the formula VLi - Cp, wherein
VI is a first antibody light chain variable region, and
C is a T-Cell Receptor β constant domain (Οβ);
b. the second polypeptide chain has the formula VL2- CL, wherein
VL2 is a second antibody light chain variable region, and
CL is a light chain κ or λ constant domain; and
c. the third polypeptide chain has a formula selected from the group consisting of
(VH1-Ca)-X1-(CH2-CH3)-X2-(VH2 -CH1),
(VH2-CHl)-(H-CH2-CH3)-X2-(VHi-Ca),
(VH!-Ca)-X2-(VH2 -CH1)-(H-CH2-CH3)> and
(VH2-CH1)-X2-(VH1-Ca)-X1-(CH2-CH3)5
wherein
YH\ is a first antibody heavy chain variable region,
Ca is a T-Cell Receptor a constant domain (Ca), Xi is a peptide linker or is absent, CH2-CJI3 are heavy chain constant domains 2 and 3,
H is a hinge region between CHI and CH2,
X2 is a peptide linker or is absent,
VH2 is a second antibody heavy chain variable region and Cm is a heavy chain constant domain 1.
A process for producing the compound of any one of Claims 1-10, comprising cultivating the cell of Claim 12 under conditions such that the compound is expressed and recovering the compound.
A compound produced by the process of Claim 13.
PCT/US2013/050436 2012-07-18 2013-07-15 Multi-specific igg-(fab)2 constructs containing t-cell receptor constant domains Ceased WO2014014796A1 (en)

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