JP5476310B2 - Monoclonal antibody binding to hGM-CSF and pharmaceutical composition comprising said antibody - Google Patents

Description

  The invention relates to a monoclonal antibody that binds to human granulocyte macrophage colony stimulating factor (also referred to as “hGM-CSF”) and neutralizes hGM-CSF activity, a composition comprising one or more of such monoclonal antibodies, And methods of using such monoclonal antibodies and compositions.

Granulocyte-macrophage colony stimulating factor (GM-CSF) has been identified as a humoral factor that promotes the proliferation of bone marrow granulocytes and macrophage progenitors and promotes the formation of granulocytes and macrophage colonies in vitro.
GM-CSF is currently known to be a broad range of cell type stimulators. GM-CSF induces the differentiation and proliferation of granulocyte-macrophage blood cells, enhances the function of antigen presenting cells, maintains the function of several types of epithelial cells, and induces the function of alveolar macrophages (Eg, enhancing surfactant catabolism, bactericidal function and Fc receptor expression). The Cytokine Handbook, 4th edition, Thomson, A .; (Author) Academic Press, 2003.
GM-CSF is a variety of diseases including 1) allergic diseases such as asthma, atopy and hay fever, 2) graft rejection and graft-versus-host disease (GVHD), and 3) autoimmune diseases such as rheumatoid arthritis. It is known to cause
For example, human GM-CSF (hGM-CSF) is overexpressed in allergic lungs and joints of rheumatoid arthritis patients, and hGM-CSF mRNA is overexpressed in allergic skin. It has also been reported that the survival of monocytes, which are inflammation-inducing cells in atopic dermatitis, is enhanced by GM-CSF production. Bratton, D.C. L. Et al., Granulocyte macrophage colony stimulating factor is involved in enhancing monocyte survival in chronic atopic dermatitis. J. et al. Clin. Invest. 95: 211-218, 1995.
Furthermore, GM-CSF has been shown to stimulate proliferation of leukemia cells. Therefore, GM-CSF is considered as a factor causing leukemia.
It would be useful to make efforts to treat diseases and conditions caused by human GM-CSF. One approach to treating such diseases and conditions is to bind to hGM-CSF and inhibit its bioactivity. This can be done, for example, by administering an anti-hGM-CSF monoclonal antibody that has high affinity and has sufficient neutralizing activity for hGM-CSF, but does not induce an immunological response.

However, the hGM-CSF inhibitory antibodies reported so far do not have sufficient neutralizing activity against hGM-CSF. Furthermore, currently available anti-hGM-CSF monoclonal antibodies are very likely to induce an unwanted immune response in the recipient. Polyclonal and monoclonal antibodies are generally obtained from laboratory animals such as mice, rabbits and goats. However, the antibodies obtained have sequences characteristic of the type of animal used for their production. When they are administered to a human, the human immune system recognizes the antibody as a foreign body, which can then result in a human anti-animal antibody response (ie, the antibody produces an antibody against itself).
In addition, long-term administration is necessary for the treatment of such diseases, which can result in problems, such as safety problems that can be caused by small amounts of impurities in the administered pharmaceutical. Antibodies with more neutralizing activity than those currently available would be valuable as therapeutic agents in terms of efficacy, safety and medical costs.

The present invention is based, at least in part, on the development by the inventors of specific anti-human GM-CSF monoclonal antibodies that are characterized by very high neutralizing activity against hGM-CSF. Surprisingly, the neutralizing activity of these antibodies is greater than can be predicted based on binding affinity for hGM-CSF. Two of these anti-hGM-CSF monoclonal antibodies are referred to herein as EV1018 and EV1019.
In one aspect, the invention provides anti-hGM-CSF antibodies and fragments thereof that bind to and neutralize hGM-CSF.

In one embodiment, an isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof is ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) within hGM-CSF. The amino acid sequence is as set forth in SEQ ID NO: 1. In one embodiment, the antibody is
(A) a heavy chain comprising a consensus V _H -CDR1 containing sequence, a consensus V _H -CDR2 containing sequence and a consensus V _H -CDR3 containing sequence,
(I) the consensus V _H -CDR1 containing sequence is FTFSX ₁ X ₂ MH (SEQ ID NO: 314), wherein X ₁ is Y or H, X ₂ is G or A;
(Ii) the consensus V _H -CDR2 containing sequence is X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), wherein each X _n is independently any natural amino acid X ₃ is L or V, X ₄ is T or I, X ₅ is Y or W, X ₆ is R or K, X ₇ is F or Y, X ₈ Is R or K;
(Iii) the consensus V _H -CDR3 containing sequence is EX _n X ₉ GX ₁₀ X _n X _n DX _n (SEQ ID NO: 316), wherein each X _n is independently any natural amino acid, X ₉ A heavy chain in which X is M or V and X ₁₀ is A or G, and (b) a light chain comprising a consensus _VL- CDR1 containing sequence, a consensus _VL- CDR2 containing sequence and a consensus _VL- CDR3 containing sequence There,
(I) the consensus V _L -CDR1-containing sequence is X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), wherein each X _n is independently any natural amino acid and X ₁₁ is H or Y,
(Ii) the consensus V _L -CDR2 containing sequence is GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein X ₁₂ is R or K, X ₁₃ is A or P;
(Iii) the consensus V _L -CDR3 containing sequence is STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319), wherein X ₁₄ is R or S and X ₁₅ is V or L, comprising a light chain, and the antibody Alternatively, the antigen-binding fragment specifically binds to hGM-CSF.

In one embodiment, the antibody is
(A) a heavy chain comprising V _H -CDR1, V _H -CDR2 and V _H -CDR3,
(I) V _H -CDR1 has an amino acid sequence selected from SYGMH (SEQ ID NO: 10) and SHAMH (SEQ ID NO: 333);
(Ii) V _H -CDR2 has an amino acid sequence selected from LTYHHGNRKFYADSVRG (SEQ ID NO: 5) and VIWHGDSKKYYADSVKG (SEQ ID NO: 334);
(Iii) a heavy chain in which V _H -CDR3 has an amino acid sequence selected from ESMGAINDN (SEQ ID NO: 6) and EWVGGTCDS (SEQ ID NO: 335), and (b) V _L -CDR1, V _L -CDR2 and V _L -CDR3 A light chain comprising
(I) V _L -CDR1 has an amino acid sequence selected from IGNNNNIGSHAVG (SEQ ID NO: 7) and SGNSSNIGSHAVG (SEQ ID NO: 330);
(Ii) V _L -CDR2 has an amino acid sequence selected from GRSPPS (SEQ ID NO: 8) and GKSPAS (SEQ ID NO: 331);
(Iii) V _L -CDR3 comprises a light chain having an amino acid sequence selected from STWDSSLSAVV (SEQ ID NO: 9) and STWDSLLSAVL (SEQ ID NO: 332), and the antibody or antigen-binding fragment thereof specifically binds to hGM-CSF To do.
For example, an antibody or antigen-binding fragment thereof described above, with a K _D of less than 400pM human GM-CSF, and more preferably binds with a K _D of less than 160 pM.
The antibodies or antigen-binding fragments described herein neutralize hGM-CSF activity, and thus, when measured at ED80 in the TF-1 proliferation assay described herein, the IC50 value of the antibody or antigen-binding fragment thereof is Less than 100 pM (eg, less than 30 pM, less than 20 pM).

In any of the embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is gamma 1 (γ ₁ ), gamma 2 (γ ₂ ), gamma 3 (γ ₃ ), or gamma 4 (γ ₄ ). Can do. The heavy chain can be selected from SEQ ID NOs: 10-33, 38-80 and 160-183 and 222-245.
In any of the embodiments, the antibody or antigen-binding fragment thereof can comprise a light chain that is a lambda light chain. In some embodiments, the lambda light chain can contain one or both of the following amino acid substitutions, R100G and A153G, as compared to the wild-type sequence. In one embodiment, the light chain can be selected from SEQ ID NOs: 34-37 and 202-221. The light chain can be a lambda light chain or a kappa light chain. In some embodiments, the heavy chain is gamma 1 (γ ₁ ), gamma 2 (γ ₂ ), gamma 3 (γ ₃ ), or gamma 4 (γ ₄ ), and the light chain is a lambda light chain. In any of the embodiments, the heavy chain is selected from the group consisting of Q3E, T97A, T97V, N95D, N95E, N95K, N95Q, N93Q / N95T, K144R, L164A and L165A as compared to the wild type sequence. One or more amino acid substitutions can be contained. In some embodiments, the antibody or antigen-binding fragment thereof is one or more selected from the group consisting of Q3E, T97A, T97V, N95D, N95E, N95K, N95Q, N93Q / N95T, K144R, L164A and L165A. Heavy chains containing amino acid substitutions and light chains containing one or both of the following amino acid substitutions, R100G and A153G.

The antibody or antigen-binding fragment thereof described herein can comprise V _H -CDR1 having the amino acid sequence SYGMH (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333). The antibody or antigen-binding fragment thereof described herein can comprise V _H -CDR2 having the amino acid sequence LTYHHGNRKFYADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVKG (SEQ ID NO: 334). The antibody or antigen-binding fragment thereof described herein can comprise V _H -CDR3 having the amino acid sequence ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS (SEQ ID NO: 335). The antibody or antigen-binding fragment thereof described herein can comprise _VL- CDR1 having the amino acid sequence IGNNNNIGSHAVG (SEQ ID NO: 7) or SGNSSNIGSYAVG (SEQ ID NO: 330). The antibody or antigen-binding fragment thereof described herein can comprise V _L -CDR2 having the amino acid sequence GRSPPS (SEQ ID NO: 8) or GKSPAS (SEQ ID NO: 331). The antibody or antigen-binding fragment thereof described herein can comprise V _L -CDR3 having the amino acid sequence STWDDSLSAVV (SEQ ID NO: 9) or STWDDSLSAVL (SEQ ID NO: 332).
In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds hGM-CSF comprises 6 different CDRs, the sequences of which are set forth as SEQ ID NOs: 4-9.

In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds hGM-CSF comprises 6 different CDRs, the sequences of which are set forth as SEQ ID NOs: 330-335.
In any of the embodiments listed herein, the antibody comprises a heavy chain and kappa that is gamma 1 (γ ₁ ), gamma 2 (γ ₂ ), gamma 3 (γ ₃ ) or gamma 4 (γ ₄ ) or A light chain that is lambda can be included.
In some embodiments, the antibodies or antigen-binding fragments described herein can further comprise a signal sequence such as those set forth in SEQ ID NOs: 324, 325, and 326.

Embodiments for wild type as well as variants thereof are included in the present invention. In some embodiments, the antibody or fragment thereof comprises a heavy chain that is a variant thereof selected from SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-80, and is selected from SEQ ID NO: 34 or SEQ ID NOs: 35-37 The light chain which is a variant thereof. In some embodiments, the heavy chain is a variant thereof selected from SEQ ID NO: 160 or SEQ ID NOs: 161-245, and the light chain is a variant thereof selected from SEQ ID NO: 202 or SEQ ID NOs: 203-221.
In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region that is a variant whose amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 152 or SEQ ID NOs: 153 to 158 and 159 And the light chain variable region wherein the amino acid sequence of the light chain variable region is a variant thereof selected from the group consisting of SEQ ID NO: 184 or SEQ ID NOs: 185-200 and 201.
In one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 152. In one embodiment, the amino acid sequence of the light chain variable region is SEQ ID NO: 184. In one embodiment, the heavy chain variable region amino acid sequence is SEQ ID NO: 152 and the light chain variable region amino acid sequence is SEQ ID NO: 184. In one embodiment, the antibody belongs to the IgG ₁ (λ) class (subclass).

In some one embodiment, the antibody or antigen-binding fragment thereof is a heavy chain variable that is a variant of which the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 348 or SEQ ID NOs: 349-362 and 363. The light chain variable region wherein the amino acid sequence of the region and the light chain variable region is SEQ ID NO: 364 or SEQ ID NO: 365.
In one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 348. In one embodiment, the amino acid sequence of the light chain variable region is SEQ ID NO: 364. In one embodiment, the amino acid sequence of the light chain variable region is SEQ ID NO: 365. In one embodiment, the antibody belongs to the IgG ₁ (λ) class (subclass).
In one embodiment, as disclosed herein, an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof that binds to hGM-CSF (hGM-CSF) and can neutralize the bioactivity of hGM-CSF. Is characterized by having a complementarity determining region (CDR) represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4-9 and SEQ ID NOs: 330-335. In some embodiments, one or more amino acids may be substituted, deleted, inserted or added in the CDR. The anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof described herein is about 14 pM of anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof when TF-1 cells are induced to proliferate by hGM-CSF. It may be characterized by inhibiting TF-1 cell growth by about 50% at a concentration. In some embodiments, the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof inhibits peripheral blood dendritic cell proliferation.
In one embodiment, the anti-hGM-CSF monoclonal antibody or antigen binding portion thereof as described herein have a high affinity at 4 × 10 ^-10 M or less of K _D values in hGM-CSF.

In some embodiments, an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof described herein belongs to the IgG ₁ (λ) class (subclass). In some embodiments, the anti-hGM-CSF monoclonal antibody of the invention is a human monoclonal antibody.
The present invention further includes a nucleic acid and a vector comprising the nucleic acid. In one embodiment, the invention is an isolated nucleic acid encoding an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof disclosed herein. In one embodiment, the nucleic acid is DNA. In one embodiment, the invention is a vector comprising DNA encoding an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof disclosed herein.
The present invention further provides a host cell comprising an expression vector comprising the DNA vector of the present invention.

The present invention also contemplates a kit comprising one or more containers containing (a) an antibody of the invention or antigen-binding fragment thereof, and (b) an antibody or antigen-binding fragment thereof.
The invention in one embodiment is an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof according to the invention for use in medicine.
The present invention provides a composition comprising an anti-hGM-CSF antibody or fragment thereof that binds and neutralizes hGM-CSF. Such a composition may be a pharmaceutical composition (eg, pharmaceutical) comprising an anti-hGM-CSF antibody and / or fragment thereof (or combination thereof) and a pharmaceutically acceptable carrier. The antibody or antigen-binding fragment thereof of any of the previous embodiments is included.

In other embodiments, the composition comprises multiple types of anti-GM-CSF antibodies, fragments or combinations thereof, each as provided herein. For example, the composition may further comprise a second isolated antibody or antigen-binding fragment thereof that binds to hGM-CSF, and thus the composition may include a plurality of types / binding agents that each bind to hGM-CSF. Including type (s) antibodies, antigen-binding fragments thereof, or combinations thereof. In some embodiments, at least one of a plurality of antibodies that bind hGM-CSF, an antigen-binding fragment thereof, or a combination thereof consists of SEQ ID NOs: 10-80, 152-245, 320-323, and 348-365. A polypeptide selected from the group.
The present invention also provides for the treatment of a disease caused by hGM-CSF comprising an anti-hGM-CSF monoclonal antibody or antigen-binding moiety according to any one of the embodiments herein and a pharmaceutically acceptable carrier. It is intended for pharmaceutical compositions suitable for use. Examples of diseases caused by overproduction of hGM-CSF include (a) allergic diseases such as asthma, atopy and hay fever (allergic rhinitis, seasonal nasal allergy), (b) graft rejection, graft pairing Host diseases (GVHD), and (c) autoimmune diseases such as rheumatoid arthritis are included.
Such compositions or veterinary pharmaceutical compositions may comprise multiple types of anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof that are specific for the same specific antigen and a pharmaceutically acceptable carrier. Good.

In some embodiments, a pharmaceutical composition or veterinary pharmaceutical composition characterized in that the plurality of types of anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof comprises two or more antibodies or antigen-binding portions thereof. The following (a) and (b):
(A) an anti-hGM-CSF monoclonal having a CDR represented by the amino acid sequence of SEQ ID NOs: 4 to 9, SEQ ID NOs: 330 to 335, SEQ ID NOs: 336 to 341, or SEQ ID NOs: 342 to 347 and specific for hGM-CSF An antibody or antigen-binding portion thereof,
(B) the anti-hGM-CSF monoclonal antibody of (a) or an antigen binding thereof having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added and specific for hGM-CSF It may be selected from parts.
Such a pharmaceutical composition or veterinary pharmaceutical composition may inhibit the proliferation of TF-1 cells by 80% or more at a concentration of about 55 pM, respectively, when the proliferation of TF-1 cells is induced by hGM-CSF. it can.

Aspects of the invention also contemplate kits that include any one of the compositions as encompassed herein and one or more containers. In some embodiments, the kit further comprises instructions. In some embodiments, the kit further comprises a label, instructions or both a label and instructions.
Also provided is the use of an antibody described herein or an antigen-binding portion thereof or a composition comprising such an antibody or fragment. Any anti-hGM-CSF antibody or antigen-binding fragment or composition thereof described herein is for producing or preparing a medicament for treating a disease or disorder associated with overexpression of hGM-CSF in a subject. This antibody or antigen-binding fragment thereof can bind to hGM-CSF and neutralize hGM-CSF activity. In some embodiments, the disease or disorder is chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, interstitial lung disease, rhinitis, arthritis and related joint diseases, psoriasis, myeloid leukemia, multiple Selected from the group consisting of multiple sclerosis.
For the described uses, the antibody or antigen-binding fragment is administered to the subject at a dose that does not exceed the maximum tolerated dose, the maximum tolerated dose being about 500 mg per dose.

One aspect of the present invention is the use of an antibody or antigen-binding fragment or composition thereof according to any one of the preceding embodiments for treating a disease or disorder associated with overexpression of hGM-CSF in a subject. This antibody or antigen-binding fragment thereof can bind to hGM-CSF and neutralize hGM-CSF activity. For example, the disease or disorder is chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, interstitial lung disease, rhinitis, arthritis and related joint diseases, psoriasis, myeloid leukemia or multiple sclerosis. May be. Such use includes administering to the subject a composition comprising an antibody or antigen-binding fragment thereof or the same as described in the present invention at a dose that does not exceed the maximum tolerated dose, About 500 mg per unit.
The invention also provides a method for treating a subject having a disease or condition associated with abnormal expression of hGM-CSF using an anti-hGM-CSF antibody or fragment thereof. This method provides an anti-hGM-CSF that neutralizes hGM-CSF activity in vivo in a subject diagnosed as at risk of developing a disease or condition associated with abnormal expression (eg, overexpression) of hGM-CSF. Administering a composition comprising the antibody or antigen-binding fragment thereof in an amount effective to obtain at least one therapeutic effect in the subject.
The present invention also provides an anti-hGM-CSF antibody-mediated hGM-, characterized by the simultaneous administration of multiple types of anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof that are specific for the same specific antigen. A method for enhancing CSF neutralizing activity is included. In some embodiments, the two or more antibodies, or antigen-binding portions thereof, are (a) and (b):
(A) an anti-hGM-CSF monoclonal having a CDR represented by the amino acid sequence of SEQ ID NOs: 4 to 9, SEQ ID NOs: 330 to 335, SEQ ID NOs: 336 to 341, or SEQ ID NOs: 342 to 347 and specific for hGM-CSF An antibody or antigen-binding portion thereof; and (b) an anti-hGM of (a) having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added and specific for hGM-CSF -Selected from CSF monoclonal antibodies or antigen-binding portions thereof.

The aspect is that two or more antibodies or antigen-binding portions thereof inhibit TF-1 cell proliferation by 80% or more at a concentration of about 55 pM each when TF-1 cells are induced to grow by hGM-CSF. And a method for enhancing activity.
The invention also provides epitopes of hGM-CSF that are recognized by the antibodies and antigen-binding fragments described herein. The epitope of hGM-CSF in the polypeptide is set forth in SEQ ID NOs: 2 and 3, which is recognized by the antibody or antigen-binding fragment thereof described in the present disclosure, and the polypeptide sequence is hGM-CSF (sequence (Based on number 1). This epitope is a discontinuous portion of human GM-CSF, and all or part of amino acid residues 77-80 of human GM-CSF (SEQ ID NO: 1) and the amino acid residue of human GM-CSF (SEQ ID NO: 1). Recognized by an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof comprising groups 95-104 and comprising 6 different CDRs as set forth in SEQ ID NOs: 4-9 or SEQ ID NOs: 330-335.
The present invention also provides the use of the epitope of the present invention. The present invention also includes methods for screening and / or identifying molecules that specifically recognize the epitopes described herein recognized by the antibodies and antigen-binding fragments thereof provided herein. Some embodiments include (i) screening a biological sample or peptide library using a probe comprising an epitope, (ii) isolating a molecule that specifically binds to the probe, and (Iii) relates to a method of identifying a molecule that binds to an epitope described herein, comprising identifying the molecule. The molecule that binds to the epitope may be, for example, an anti-hGM-CSF monoclonal antibody or an antigen-binding fragment thereof. Some embodiments contemplate probes that further include a detectable marker. In some embodiments, the method also incorporates an immobilized probe.

Also provided are methods or processes for producing anti-hGM-CSF antibodies and antigen-binding fragments thereof. The present invention provides a method for producing an anti-hGM-CSF monoclonal antibody that binds to hGM-CSF and an antigen-binding fragment thereof in a host cell, wherein the antibody or the antigen-binding fragment thereof is at least a consensus V _H -CDR1-containing sequence. , Consensus V _H -CDR2 containing sequences, consensus V _H -CDR3 containing sequences, consensus V _L -CDR1 containing sequences, consensus V _L -CDR2 containing sequences and consensus V _L -CDR3 containing sequences. This method
(I) Consensus V _H -CDR1 containing sequence, Consensus V _H -CDR2 containing sequence, Consensus V _H -CDR3 containing sequence, Consensus V _L -CDR1 containing sequence, Consensus V _L -CDR2 containing sequence and Consensus V _L -CDR3 containing Obtaining a host cell comprising at least one DNA sequence encoding the sequence, comprising:
(A) the consensus V _H -CDR1 containing sequence is FTFSX ₁ X ₂ MH (SEQ ID NO: 314), wherein X ₁ is Y or H, X ₂ is G or A;
(B) the consensus V _H -CDR2 containing sequence is X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), wherein each X _n is independently any natural amino acid X ₃ is L or V, X ₄ is T or I, X ₅ is Y or W, X ₆ is R or K, X ₇ is F or Y, X ₈ Is R or K;
(C) the consensus V _H -CDR3 containing sequence is EX _n X ₉ GX ₁₀ X _n X _n DX _n (SEQ ID NO: 316), wherein each X _n is independently any natural amino acid, X ₉ Is M or V, X ₁₀ is A or G,
(D) the consensus V _L -CDR1 containing sequence is X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), wherein each X _n is independently any natural amino acid and X ₁₁ is H or Y,
(E) the consensus V _L -CDR2 containing sequence is GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein X ₁₂ is R or K, X ₁₃ is A or P;
(F) the step wherein the consensus V _L -CDR3 containing sequence is STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319), wherein X ₁₄ is R or S, and X ₁₅ is V or L;
(Ii) expressing the DNA in a host cell, and (iii) culturing the host cell under conditions suitable for expression of the DNA and production of the antibody or antigen-binding fragment thereof.

In some embodiments, the at least one DNA encodes a heavy chain or part thereof and a light chain or part thereof, wherein the heavy chain or part thereof is SEQ ID NO: 10-33, 38-80, 152. Selected from the group consisting of ~ 183, 222-245, 348-362 and 363, wherein the light chain or part thereof has a sequence selected from the group consisting of SEQ ID NOs: 34-37, 184-221, 364 and 365 .
The process can further include isolating the antibody or antigen-binding fragment thereof.
The process can further comprise preparing a composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

Also provided are vectors comprising DNA encoding at least a portion of an antibody or antigen-binding fragment thereof described herein. Also included are host cells that express such vectors.
In some embodiments, the vector comprises DNA encoding _VH- CDR1, _VH- CDR2, and _VH- CDR3;
V _H -CDR1 is SYGMH (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333);
V _H -CDR2 is LTYHHGNRKFYADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVKG (SEQ ID NO: 334);
V _H -CDR3 is ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS (SEQ ID NO: 335).
In some embodiments, the vector comprises DNA encoding _VL- CDR1, _VL- CDR2, and _VL- CDR3;
V _L -CDR1 is IGNNNNIGSHAVG (SEQ ID NO: 7) or SGNSSNIGSHAVG (SEQ ID NO: 330);
V _L -CDR2 is GRPSPPSG (SEQ ID NO: 8) or GKSPASG (SEQ ID NO: 331);
V _L -CDR3 is STWDSSLSAVV (SEQ ID NO: 9) or STWDDSLSAVL (SEQ ID NO: 332).

  In some embodiments of the invention, the isolated DNA is characterized in that it encodes an amino acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 4-9. Encodes an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof that can bind to hGM-CSF and neutralize the bioactivity of hGM-CSF. In some embodiments, the isolated DNA encodes an amino acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 330 to 335, wherein the isolated DNA is hGM- It encodes an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof that can bind to CSF and neutralize the physiological activity of hGM-CSF. Isolated DNA may be able to hybridize with the aforementioned DNA under stringent conditions. Vectors incorporating any such DNA, as well as host cells that express the recombinant expression vector (s) are included in embodiments of the invention.

It is a figure which shows the flowchart of isolation of an antibody producing cell clone. It is a figure which shows the scheme of the affinity analysis of an anti-GM-CSF monoclonal antibody. It is a figure which shows evaluation of the inhibitory effect of an anti-GM-CSF monoclonal antibody with respect to the proliferation of the peripheral blood dendritic cell in presence of GM-CSF. It is a figure which shows evaluation of the inhibitory effect of an anti-GM-CSF monoclonal antibody with respect to the proliferation of TF-1 cell in yeast hGM-CSF (Leukine) presence. It is a figure which shows evaluation of the inhibitory effect of an anti-GM-CSF monoclonal antibody with respect to the proliferation of TF-1 cell in colon_bacillus | E._coli hGM-CSF (Peprotech) presence. It is a figure which shows evaluation of the inhibitory effect of the combined use of two anti-GM-CSF monoclonal antibodies with respect to the proliferation of TF-1 cell in yeast hGM-CSF (Leukine) presence. It is a figure which shows evaluation of the inhibitory effect of the combination use of two anti-GM-CSF monoclonal antibodies with respect to the proliferation of TF-1 cell in colon_bacillus | E._coli hGM-CSF (Peprotech) presence. FIG. 2 shows two graphs representing dose-dependent inhibition of human GM-CSF (8A) and rhesus monkey GM-CSF (8B). FIG. 5 is a bar graph depicting myeloperoxidase (MPO) activity measured in bronchoalveolar fluid (BALF) pellets from mice exposed to cigarette smoke. CS: mice treated with vehicle (300 μl of vehicle ip on days 1 and 3), no exposure to tobacco smoke. + CS, isoform AB: 300 μl of isoform antibody i. p. Exposed to cigarette smoke. + CS, anti-GM-CSF AB: on days 1 and 3, 300 μl of anti-GM-CSF antibody i. p. Exposed to cigarette smoke. N = 10 per group, mean ± SEM, ^* p <0.05, ^*** p <0.001. FIG. 4 shows a pair of bar graphs representing cytokine measurements in BALF supernatant. Left: Measurement of MCP-1 level. Right: MIP-1α level measurement. CS: mice treated with vehicle (300 μl of vehicle ip on days 1 and 3), no exposure to tobacco smoke. + CS isoform AB: 300 μl of isoform antibody i. p. Exposed to cigarette smoke. + CS, anti-GM-CSF AB: on day 1 and day 3 with 300 μl of anti-GM-CSF antibody i. p. Treated mice, exposed to cigarette smoke. N = 10 per group, mean ± SEM, ^* p <0.05, ^*** p <0.001.

  As described herein, anti-hGM-CSF monoclonal antibodies were obtained from blood-producing antibody-producing cells of patients with unexplained alveolar proteinosis. In one embodiment, the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof of the present invention is obtained from antibody-producing cells derived from blood of an alveolar proteinosis patient of unknown cause. Accordingly, the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof in one embodiment of the present invention is a fully human monoclonal antibody. Thus, when an antibody preparation is administered into the human body, the risk of causing immunogenicity is minimized.

The problems to be solved by the present invention are as follows. The present invention includes GM-CSF binding monoclonal antibodies and GM-CSF binding fragments thereof that exhibit improved ability or activity neutralization against hGM-CSF and at least one or one form of such monoclonal antibodies or fragments thereof. Compositions and methods for treating symptoms or diseases associated with overexpression of hGM-CSF (in individuals suffering from symptoms, diseases or disorders, hGM-CSF levels are higher in individuals suffering from symptoms, diseases or disorders) Note that providing high).
The anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof described in this disclosure specifically binds to hGM-CSF whose abnormal expression can cause various diseases, and suppresses or eliminates (neutralizes) its physiological activity. To do. The anti-hGM-CSF monoclonal antibody exhibits a higher neutralizing activity (against hGM-CSF) than the existing anti-hGM-CSF monoclonal antibody. In particular, the anti-hGM-CSF monoclonal antibody of the present invention, which is a human monoclonal antibody, is not immunogenic and does not induce an immune response.

  By “specifically binds” is meant high binding activity and / or high affinity binding to an epitope of a specific polypeptide of an antibody, eg, a protein such as the human GM-CSF protein. Since the specific polypeptide of interest binds more strongly to an epitope fragment of a protein such as, for example, GM-CSF, antibody binding to an epitope on this specific polypeptide can be any other epitope, particularly related It is preferred that the antibody bind stronger than the same antibody, or an epitope that may be present on a molecule in the same sample, and thus by adjusting the binding conditions, the antibody may become an epitope site or fragment of the desired protein, e.g. , Binds almost exclusively to epitope fragments exposed to GM-CSF treatment and not to those exposed to related factors of the same subfamily. Isolated antibodies of the present invention are preferably immunoreactive with a particular species and immunospecific. For example, the antibodies of the invention recognize human GM-CSF and can recognize rhesus monkey GM-CSF and / or marmoset GM-CSF, but generally do not recognize the mouse counterpart. The antibodies and antigen-binding fragments thereof disclosed in this aspect of the present invention are such that when the epitope of the antigen is observed in a natural conformation or by structural studies, a discontinuous portion of the antigen is very small within the whole antigen (eg, hGM-CSF). In a conformation that is substantially close to nature so that they are brought together and thereby together form an epitope (eg, a “pocket” on the antigen surface) that is recognized by the antibodies of the invention. Please understand that you recognize. Thus, the antibodies or antigen-binding fragments thereof described in this aspect of the invention do not cross-react with proteins that are not GM-CSF that contain both the polypeptide sequences that form the epitope as described above. It should be understood that in the case of antibody-antigen binding, the binding is considered “specific” due to the nature of the interaction. Thus, “an antibody that binds to GM-CSF” is specifically interpreted as binding to an antigen (ie, GM-CSF).

  As used herein, the term “antibody” refers to an immunoglobulin molecule in which four polypeptide chains, two heavy chains, and two light chains are interactively linked by disulfide bonds. Monoclonal antibodies can be made by monoclonal or recombinant techniques. This technique is a well-known technique. The terms “antigen-binding portion” and “antigen-binding fragment” are used interchangeably herein, preferably any antibody that exhibits specific antigen-binding activity against an antigen (eg, hGM-CSF). Represents a short variant. In particular, the fragment of the present invention is a fragment that binds to hGM-CSF at the same epitope as each monoclonal antibody. The fragment may be, for example, a Fab fragment or any other fragment that exhibits the aforementioned properties. In addition, antibody fragments further include short chain antibodies (scFV), bispecific antibodies and multispecific antibodies that incorporate the variable region and complementarity determining region (CDR) of the antibody.

  As used herein, “physiological activity” is synonymous with biological activity. Thus, “bioactivity of hGM-CSF” is the biological function of hGM-CSF determined or measured in an in vivo or cell-based assay, eg, an assay described herein. . More specifically, the physiological activity of hGM-CSF can be measured by a cell proliferation assay. Appropriate cell lines known to respond to GM-CSF are used. Usually, for human cell lines, the human erythroleukemia cell line, TF-1, is used to perform proliferation assays. TF-1 is a cell line established from patients with erythroleukemia. This assay is based on the inhibition of GM-CSF stimulated cell proliferation of TF-1 cells and is well established in the art. Other cell lines may be appropriate. One skilled in the art will be able to adapt appropriate assay systems or conditions.

The TF-1 proliferation assay is performed in the presence of various concentrations of wild type recombinant human GM-CSF (referred to as rhGM-CSF). Based on these data, a dose-dependent curve can be obtained. The concentration of GM-CSF required to elicit 80% of the maximum biological response of proliferation in TF-1 cells are herein defined as ED ₈₀ or ED80. Therefore, ED80 represents “80% effective amount” of the maximum response. Similarly, the concentration of GM-CSF required to show the maximal response half in biological assay is expressed as ED ₅₀ or ED50. Usually, the ED50 value of GM-CSF is said to be 0.02-1 ng / ml as measured in a cell proliferation assay using a human GM-CSF dependent TF-1 cell line.

This assay system or any other similar assay system can be used to identify or measure the activity of inhibitors or antagonists of hGM-CSF. For example, a proliferation assay system can be used to measure the neutralizing ability of antibodies to hGM-CSF. Other examples of assays for measuring biological GM-CSF function include induction of IL-8 secretion assay and surface CD11b / Mac1 assay.
The terms “neutralize”, “neutralize”, “neutralize” and the like mean inhibition of the biological activity of a bioactive factor, such as hGM-CSF. Thus, an antibody against hGM-CSF with neutralizing ability or neutralizing activity binds to hGM-CSF in a biological system and the biological activity of hGM-CSF, eg, biological system Means, eg, can antagonize or counter the proliferative effect of hGM-CSF in TF-1 cells, thereby substantially inhibiting or eliminating the biological activity of hGM-CSF .
Terms such as “inhibitory effect”, “inhibition”, “inhibitory ability” and “inhibition” mean a physiological activity or 5 to 100% suppression of physiological activity caused by a target antigen, but are limited only to its source Not. For therapeutic purposes, ideally, inhibition of hGM-CSF bioactivity should be between 50% and 100%.

In general, the IC ₅₀ is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This quantitative criterion determines which specific drug or other drug (inhibitor) is used to half inhibit a given biological process (or component of the process, ie, enzyme, cell, cell receptor or microorganism) Only show what is needed. In other words, it is the half-maximal (50%) inhibitory concentration (IC) of the substance (50% IC, or IC ₅₀ ). Usually used in pharmacological studies as a measure of antagonist efficacy. According to US Food and Drug Administration guidelines, the IC ₅₀ represents the drug concentration required to inhibit 50% in vitro.
Thus, the IC ₅₀ of neutralizing antibodies to hGM-CSF can be determined by generating dose response curves and examining the effect of various concentrations of neutralizing antibody on reversing the proliferation activity of hGM-CSF. IC ₅₀ values can be calculated for a given antagonist by measuring the concentration required to inhibit half of the maximal biological response of the agonist.
According to various embodiments, the IC50 value of the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof is less than 20 pM, less than 25 pM, less than 30 pM, less than 40 pM, as measured by ED80 in each case in a TF-1 proliferation assay, Less than 50 pM, less than 60 pM, less than 70 pM, less than 80 pM, less than 90 pM or less than 100 pM. In certain embodiments, the IC50 value of the antibody or antigen-binding fragment thereof is less than 20 pM, less than 25 pM, less than 30 pM, or less than 40 pM as measured by ED80 in a TF-1 proliferation assay.

The anti-hGM-CSF monoclonal antibody in the present disclosure comprises an immunoglobulin molecule comprising two heavy chains and two light chains. The heavy chains are respectively abbreviated heavy chain variable region (appropriately abbreviated as “HCVR” or “V _H ”) and heavy chain constant region (heavy chain constant regions are abbreviated as “CH1”, “CH2” and “CH3”). With 3 domains). Each light chain is a light chain variable region (abbreviated appropriately as “LCVR” or “V _L ”) and a light chain constant region (the light chain constant region has one domain abbreviated as “C _L ”). including. HCVR and LCVR are particularly important for antibody binding specificity.
The amino acid sequence of the variable region is responsible for most of the interactions between the antibody and the antigen. Thus, when constructing an expression vector that contains a variable region sequence derived from a natural antibody within a framework sequence derived from a different antibody with different properties, the resulting vector will recombine to mimic the properties of the natural antibody. Antibodies can be expressed. Thus, it is not necessary to obtain the complete sequence of a particular antibody when making a complete recombinant antibody with similar binding properties as the original antibody. Sometimes the heavy and light chain subsequences encompassing the variable region may be sufficient to achieve that purpose.
The antibody interacts with the target antigen mainly through amino acid residues of LCVR and HCVR. Therefore, the amino acid sequence of the variable region is more diverse in the antibody than sequences other than the variable region. HCVR and LCVR are further subdivided into relatively stable “framework regions (FR)” and hypervariable “complementarity determining regions (CDRs)”. HCVR and LCVR are each composed of 3 CDRs and 4 FRs. The order is FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the amino terminus to the carboxy terminus.

Methods for determining or predicting amino acid residues corresponding to each of the antibody structural domains, including the hypervariable regions, eg, CDRs, are well known in the art. Some of the commonly used methods include Chothia, AbM and Kabat.
In general, an antibody binds to an antigen with an affinity of at least about 1 × 10 ⁻⁷ M and binds to a specific antigen with an affinity that is at least 2-fold higher compared to a non-specific antigen (eg, BSA, casein). Join.
The term “high affinity” for a particular IgG antibody means that the binding affinity of the antibody is at least about 1 × 10 ⁻⁷ M, preferably at least about 1 × 10 ⁻⁸ M, more preferably at least 1 × 10 ^−9. M, even more preferably means at least 1 × 10 ⁻¹⁰ M.
The definition of “high affinity” varies among antibody isotypes. For example, an IgM isotype is defined as having a “high affinity” when the binding affinity is at least 1 × 10 ⁻⁷ M.

Antibodies of the Invention As described herein, the present invention is an anti-human GM- that has a higher neutralizing ability against hGM-CSF than existing monoclonal antibodies against hGM-CSF (than currently available monoclonal antibodies). The present invention relates to a CSF (anti-hGM-CSF) monoclonal antibody and fragments thereof. One type or type of anti-hGM-CSF monoclonal antibodies or fragments thereof described herein can be used or administered.

  Alternatively, multiple types or types of anti-hGM-CSF monoclonal antibodies can be used or administered together. The inventors have found that when multiple types or types of anti-GM-CSF monoclonal antibodies are used together, they provide high neutralizing activity (eg, high cell growth inhibitory activity). Further, one or more types or types of anti-hGM-CSF monoclonal antibodies or fragments thereof are used or administered in combination with one or more other therapeutic agents that are not antibodies, such as small molecules, RNAi or peptides. be able to.

  In one aspect, an isolated monoclonal antibody or antigen-binding fragment thereof that specifically recognizes and binds to hGM-CSF is provided. Binds to hGM-CSF (hGM-CSF binding to hGM-CSF and anti-hGM-CSF monoclonal antibody binding to antigen binding fragments thereof) and exhibits neutralizing ability or neutralizing activity against the biological activity of hGM-CSF Isolated anti-hGM-CSF monoclonal antibodies and antigen-binding fragments thereof are described herein. In one embodiment, an anti-hGM-CSF monoclonal antibody and antigen-binding fragment thereof that bind to hGM-CSF and exhibit neutralizing ability or activity against hGM-CSF are isolated, the monoclonal antibody and antigen-binding fragment thereof being hGM- Recognizes ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in CSF (SEQ ID NO: 1). Isolated anti-hGM-CSF monoclonal antibodies and antigen-binding fragments thereof that bind to hGM-CSF that recognize ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF are It is useful for treating and preventing diseases, conditions and disorders that exhibit sum ability or activity and are associated with overexpression of hGM-CSF. A specific embodiment is an isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof that binds hGM-CSF, wherein the antibody or antigen-binding fragment is an amino acid at positions 77-80 in hGM-CSF. Recognize the following defined hGM-CSF discontinuous epitope (SEQ ID NO: 1) corresponding to ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) at positions 95-104 in hGM-CSF. The isolated anti-hGM-CSF monoclonal antibodies and antigen-binding fragments thereof described herein specifically bind to hGM-CSF. These do not cross-react with proteins that are proteins other than GM-CSF.

More specifically, described herein are monoclonal antibodies that have the ability to specifically bind to hGM-CSF and neutralize the biological activity of hGM-CSF. The four such antibodies described herein are designated EV1018, EV1019, EV1003 and EV1007, as summarized in Tables 1 and 2 in the examples. Therefore, EV1018, EV1019, EV1003 and EV1007 can bind to hGM-CSF and neutralize the proliferation promoting effect of hGM-CSF. As shown in more detail, EV1018 and EV1019 antibodies recognize the same or substantially overlapping epitopes on hGM-CSF protein.
An anti-hGM-CSF monoclonal antibody or an antigen-binding portion thereof having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in a complementarity determining region (CDR) Is further encompassed by.

In a further aspect, there is an isolated deoxyribonucleic acid (DNA) encoding an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof that can bind to hGM-CSF and neutralize the biological activity of hGM-CSF. Thus, an isolated DNA is provided that encodes an amino acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 4-9.
One embodiment of the present invention is an anti-hGM-CSF monoclonal antibody or antigen-binding moiety that binds to hGM-CSF (hGM-CSF) and can neutralize the biological activity of hGM-CSF (hGM-CSF). The present invention relates to an anti-hGM-CSF monoclonal antibody or antigen-binding portion having a complementarity determining region (CDR) represented by at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 9.

Another embodiment of the invention is an anti-hGM-CSF monoclonal antibody or antigen-binding portion that can bind to hGM-CSF and neutralize the biological activity of hGM-CSF, comprising SEQ ID NOs: 330-335. The present invention relates to an anti-hGM-CSF monoclonal antibody or an antigen-binding portion having a complementarity determining region (CDR) represented by at least one amino acid sequence selected from the group consisting of
In the present invention, an anti-hGM-CSF monoclonal antibody having a complementarity determining region (CDR) represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4-9, and SEQ ID NOs: 330-335 Anti-hGM-CSF monoclonal antibodies having a complementarity determining region (CDR) represented by one or more amino acid sequences selected from the group are considered as different types of anti-hGM-CSF monoclonal antibodies.

A monoclonal antibody or antigen-binding portion thereof in which one or more amino acids have been deleted, substituted, inserted or added in a complementarity determining region (CDR), is a biological antibody that binds specifically to hGM-CSF and whose biological As long as the activity is neutralized, it is included in the present invention.
The present invention includes a monoclonal antibody or antigen-binding portion thereof that can bind to hGM-CSF and neutralize the physiological activity of hGM-CSF, and the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof comprises:
(A) a light chain (L chain) CDR1 having the amino acid sequence of SEQ ID NO: 7,
(B) a light chain (L chain) CDR2 having the amino acid sequence of SEQ ID NO: 8, and (c) a light chain (L chain) CDR3 having the amino acid sequence of SEQ ID NO: 9
Having a CDR selected from the group consisting of

Similarly, the present invention includes a monoclonal antibody or antigen-binding portion thereof that can bind to hGM-CSF and neutralize the physiological activity of hGM-CSF, wherein the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof comprises:
(D) a light chain (L chain) CDR1 having the amino acid sequence of SEQ ID NO: 330,
(E) a light chain (L chain) CDR2 having the amino acid sequence of SEQ ID NO: 331, and (f) a light chain (L chain) CDR3 having the amino acid sequence of SEQ ID NO: 332
Having a CDR selected from the group consisting of
The following anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof, wherein the light chain complementarity determining region (CDR) described above is modified by deletion, substitution, insertion or addition of one or more amino acids A CSF monoclonal antibody or antigen-binding portion thereof is further incorporated into the present invention when it specifically binds to hGM-CSF and neutralizes its bioactivity.

The present invention includes an anti-hGM-CSF monoclonal antibody or an antigen-binding portion thereof capable of binding to hGM-CSF and neutralizing the physiological activity of hGM-CSF, wherein the anti-hGM-CSF monoclonal antibody or the antigen-binding portion thereof is ,
(A) a heavy chain (H chain) CDR1 having the amino acid sequence of SEQ ID NO: 4,
(B) heavy chain (H chain) CDR2 having the amino acid sequence of SEQ ID NO: 5, and (c) heavy chain (H chain) CDR3 having the amino acid sequence of SEQ ID NO: 6
Having a CDR selected from the group consisting of

Similarly, the present invention includes a monoclonal antibody or antigen-binding portion thereof that can bind to hGM-CSF and neutralize the physiological activity of hGM-CSF, and the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof comprises:
(A) a heavy chain (H chain) CDR1, having the amino acid sequence of SEQ ID NO: 333,
(B) heavy chain (H chain) CDR2 having the amino acid sequence of SEQ ID NO: 334, and (c) heavy chain (H chain) CDR3 having the amino acid sequence of SEQ ID NO: 335
Having a CDR selected from the group consisting of
The following anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof is further incorporated into the present invention when it specifically binds to hGM-CSF and neutralizes its bioactivity.
An anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof, wherein the previously described heavy chain complementarity determining region (CDR) is modified by deletion, substitution, insertion or addition of one or more amino acids.

GM-CSF epitope The epitope of hGM-CSF, a discontinuous segment of human GM-CSF, is also the subject of this specification, which epitope is amino acid residues 77-80 of human GM-CSF (SEQ ID NO: 1) and human 6 amino acids residues 95-104 of GM-CSF (SEQ ID NO: 1), defined by the polypeptide sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3, and set forth in SEQ ID NO: 4-9 or SEQ ID NO: 330-335 Recognized by anti-hGM-CSF monoclonal antibodies or antigen-binding fragments thereof containing different CDRs.
As described in more detail in the examples, epitopes of hGM-CSF recognized by monoclonal antibodies EV1018 and EV1019 were identified by epitope mapping studies. It is found that both EV1018 and EV1019 recognized the same (or substantially overlapping) site of the hGM-CSF antigen corresponding to amino acid residues 77-80 and 95-104 determined according to GenBank accession number NP_000749 It was. Structural studies indicate that the two segments are discontinuous in the primary sequence, but approach when the two segments of the polypeptide fold, forming a conformational epitope together. Consistent with the observation that both EV1018 and EV1019 recognize the same or substantially overlapping epitopes, analysis of the CDR sequences of EV1018 and EV1019 shows significant similarity. The peptide sequences of the CDRs of these two antibodies are compared to obtain the following consensus sequences (eg, formulas) that represent overlapping (eg, common) residues that contain each of the CDRs.

For V _H -CDR1: FTFSX ₁ X ₂ MH (SEQ ID NO: 314), wherein X ₁ is Y or H and X ₂ is G or A;
Regarding V _H -CDR2: X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), wherein each X _n is independently any natural amino acid, and X ₃ is L Or V, X ₄ is T or I, X ₅ is Y or W, X ₆ is R or K, X ₇ is F or Y, and X ₈ is R or K;

For V _H -CDR3: EX _n X ₉ GX ₁₀ X _n X _n DX _n (SEQ ID NO: 316), wherein each X _n is independently any natural amino acid, X ₉ is M or V; X ₁₀ is A or G;
For V _L -CDR1: X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), wherein each X _n is independently any natural amino acid and X ₁₁ is H or Y;

For V _L -CDR2: GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein X ₁₂ is R or K, X ₁₃ is A or P; and for V _L -CDR3: STWDSX ₁₄ LSAV X ₁₅ ( SEQ ID NO: 319), wherein X ₁₄ is R or S, and X ₁₅ is V or L.

In each of the consensus sequence formulas, the amino acid residues containing the corresponding CDRs are shown in conventional single letter form. The same residues shared by both EV1018 and EV1019 are shown. Accordingly, another embodiment herein is an isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof that binds to hGM-CSF and exhibits neutralizing ability or activity against hGM-CSF, wherein antibody or antigen-binding fragment thereof, (a) (i) X ₁ is Y or H, X ₂ is G or a consensus V _H -CDR1 containing sequence FTFSX ₁ X ₂ MH (SEQ ID NO: 314), ( ii) each X _n is independently any natural amino acid, X ₃ is L or V, X ₄ is T or I, X ₅ is Y or W, X ₆ is R or K A consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), wherein X ₇ is F or Y and X ₈ is R or K; and (iii) each X _n is Germany It is any naturally occurring amino acid, a X ₉ is M or V, consensus V _H -CDR3 containing sequence X ₁₀ is A or _{G EX n X 9 GX 10 X} n X n DX n ( SEQ ID NO: 316) Or a portion of the heavy chain in the case of an antibody fragment, and (b) (i) a consensus V _L -CDR1 where each X _n is independently any natural amino acid and X ₁₁ is H or Y Containing sequence X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), (ii) Consensus V _L -CDR2 containing sequence GX ₁₂ SPX ₁₃ SG (X ₁₂ is R or K and X ₁₃ is A or P) SEQ ID NO: 318), and (iii) X ₁₄ is R or S, the light chain comprising a consensus V _L -CDR3 containing sequence STWDSX ₁₄ LSAVX ₁₅ X ₁₅ is V or L (SEQ ID NO: 319) or anti, For fragments comprise a portion of the light chain, the antibody or fragment thereof recognizes ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF. In a specific embodiment, the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof binds to a discontinuous epitope in ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF (SEQ ID NO: 1). To do.

Another embodiment described herein is an isolated anti-hGM-CSF monoclonal antibody that binds hGM-CSF or an antigen-binding fragment thereof that binds hGM-CSF, wherein the antibody or fragment thereof is ( a) (i) a consensus V _H -CDR1-containing sequence FTFSX ₁ X ₂ MH (SEQ ID NO: 314) wherein X ₁ is Y or H and X ₂ is G or A, (ii) each X _n is independently Any natural amino acid, X ₃ is L or V, X ₄ is T or I, X ₅ is Y or W, X ₆ is R or K, X ₇ is F or Y A consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), wherein X ₈ is R or K, and (iii) each X _n is independent Any natural amino acid, X ₉ is M Or a V, when X ₁₀ is heavy or antibody fragment, including consensus V _H -CDR3 containing sequence is A or _{G EX n X 9 GX 10 X} n X n DX n ( SEQ ID NO: 316) a heavy chain And (b) (i) a consensus V _L -CDR1-containing sequence X _n GNX _n X _n NIGSX ₁₁ AVG (sequence) wherein each X _n is independently any natural amino acid and X ₁₁ is H or Y 317), (ii) a consensus _VL- CDR2-containing sequence GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein X ₁₂ is R or K and X ₁₃ is A or P, and (iii) X ₁₄ is R or S, and if X ₁₅ is a light chain or an antibody fragment, including consensus V _L -CDR3 containing sequence STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319) is V or L comprises a portion of the light chain, the Body or fragments thereof, to hGM-CSF, following 400 pM, in another embodiment attached with a K _D of less than 160 pM, K _D is determined in accordance with the techniques described in Example 11.

In another embodiment, the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof is the anti-hGM-CSF monoclonal antibody or antigen-binding anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof is a peripheral blood dendritic cell proliferation. It is characterized by inhibiting. Thus, a further embodiment is an isolated anti-hGM-CSF monoclonal antibody that binds hGM-CSF or an antigen-binding fragment thereof that binds hGM-CSF, wherein the anti-hGM-CSF monoclonal antibody or fragment thereof is A chain or a portion thereof and a light chain or a portion thereof. In the case of a heavy chain or antibody fragment, a portion of the heavy chain can include V _H -CDR1, V _H -CDR2 and V _H -CDR3. In some embodiments, the consensus sequence is represented by the formula FTFSX ₁ X ₂ MH (SEQ ID NO: 314) comprising V _H -CDR1, wherein X ₁ is Y or H and X ₂ is G or A. . Similarly, each X _n is independently any natural amino acid, X ₃ is L or V, X ₄ is T or I, X ₅ is Y or W, and X ₆ is R or K. Wherein X ₇ is F or Y and X ₈ is R or K, the formula X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315) is V _H -CDR2 Wherein each X _n is independently any natural amino acid, X ₉ is M or V, and X ₁₀ is A or G. EX _n X ₉ GX ₁₀ X _n X _n DX _n (SEQ ID NO: 316) represents a consensus sequence comprising V _H -CDR3. In the case of a light chain or antibody fragment, the light chain or a portion thereof can comprise V _L -CDR1, V _L -CDR2 and V _L -CDR3. In such embodiments, the formula X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), wherein each X _n is independently any natural amino acid and X ₁₁ is H or Y is V _L -CDR1 Formula GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein X ₁₂ is R or K and X ₁₃ is A or P, represents a consensus sequence comprising V _L -CDR2 and is represented by the formula STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319) represents a consensus sequence comprising V _L -CDR3.

  In any of the embodiments, the antibody or fragment thereof neutralizes hGM-CSF activity. In a specific embodiment, the anti-hGM-CSF monoclonal antibody or fragment thereof is determined as described in the TF-1 proliferation assay performed in each case with ED80 and in Example 12. HGM-CSF activity is neutralized to have an IC50 value in the growth inhibition assay of less than 20 pM, less than 30 pM, less than 40 pM, less than 50 pM, less than 60 pM, less than 70 pM, less than 80 pM, less than 90 pM, 90 pM or less than 100 pM. In a specific embodiment, the claimed anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof has an IC50 value that is less than 40 pM or less than 30 pM as determined in the ED-1 TF-1 proliferation assay. In a specific embodiment, the claimed anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof has an IC50 value that is less than 25 pM or less than 20 pM as determined in the ED80 TF-1 proliferation assay. In a further embodiment, the invention provides an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof, wherein the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof proliferates TF-1 cells by induction of hGM-CSF. Then, it is characterized by inhibiting the growth of about 50% TF-1 cells at a concentration of about 14 pM.

  As demonstrated in more detail in the examples, both EV1018 and EV1019 are effective in neutralizing the bioactivity of hGM-CSF. Since these antibodies substantially share the same conformational epitope as previously described, the epitope can be used to screen for additional molecules that recognize the epitope, such as monoclonal antibodies or fragments thereof. it can. Accordingly, the present invention also contemplates screening methods in which the epitope of hGM-CSF is defined by the polypeptide sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3 and is recognized by EV1018, EV1019 or variants thereof. Such methods include (i) screening a biological sample or peptide library using a probe comprising an epitope, (ii) isolating molecules that specifically bind to the probe, and ( iii) including identifying the molecule. In one embodiment, the method further comprises generating a molecule. In some embodiments, the molecule identified using this method is an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof. In some cases, the probe can further include a detectable marker including, but not limited to, a label, a dye, an affinity tag, and the like. According to some embodiments, the probe can be immobilized, eg, for screening binding molecules. Screening methods are generally available and are well known to those skilled in the art.

In any of the embodiments described herein, the antibody or antigen-binding fragment thereof may be any subtype of immunoglobulin, such as an IgG class such as IgG ₁ , IgG ₂ , and IgG ₄ . Thus, in any of the embodiments described herein, the heavy chain is a gamma such as, for example, gamma 1 (γ ₁ ), gamma 2 (γ ₂ ), gamma 3 (γ ₃ ) or gamma 4 (γ ₄ ). It may be a heavy chain of class. In any of the previously described embodiments, the heavy chain can be selected from the group consisting of the polypeptides set forth in SEQ ID NOs: 10-33, 38-80 and 152-245, and 348-363 (heavy chain amino acids). Residues are selected from these). In some embodiments, a portion of the heavy chain can be used, including the variable region of the heavy chain. Non-limiting examples of heavy chain variable regions described in this disclosure are provided in SEQ ID NOs: 152-159 and 348-363.

  In anti-hGM-CSF monoclonal antibodies and antigen-binding fragments thereof, the light chain may be a lambda light chain, or a portion thereof, or a kappa light chain, or a portion thereof. In a specific embodiment, the lambda light chain contains one or both of the following amino acid substitutions: R100G and A153G (containing substitution R100G, substitution A153G, or both substitution R100G and substitution A153G). Substitutions in the amino acid sequence of an antibody chain are described herein by reference to the appropriate reference amino acid sequence, and designations for positions 100 and 153 in any light chain or fragment thereof are such that the amino acid position is SEQ ID NO: 34. When compared with the sequence in the sequence, it corresponds to the amino acid position in SEQ ID NO: 35 in which R is replaced with G and A is replaced with G. Therefore, the present invention is determined by comparison between SEQ ID NO: 35 and SEQ ID NO: 34. It must be noted that For example, in the foregoing case, where the lambda light chain may contain one or both of the amino acid substitutions described, the reference amino acid sequence is EV1018 light chain (EV1018 wild type, SEQ ID NO: 34). R100G indicates that R (arginine) at position 100 in the EV1018 light chain is replaced by G (glycine), and A153G replaces A (alanine) at position 153 in the EV1018 light chain by G (glycine) It shows that.

In a specific embodiment, the light chain is selected from the group consisting of the polypeptides set forth in SEQ ID NOs: 34-37 and 184-221 and 364-365, the amino acid residues of which are represented by these. Examples of light chain variable regions described herein are provided in SEQ ID NOs: 184-201 and 364-365.
The light chain contains one or more combinations of these amino acid substitutions and other amino acid substitutions that do not substantially impair the ability of the antibody or antigen-binding fragment thereof to bind to hGM-CSF and neutralize its activity. Can be included. In one embodiment, the light chain comprises amino acid substitutions at one or both of positions 100 and 153 such that glycosylation is not possible at one or both of these positions.

The heavy chain of an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof that binds to hGM-CSF is one or more such as T97A, T97V, N95D, N95E, N95K, N95Q, N93Q / N95T, K144R, L164A, and L165A. Multiple amino acid substitutions can be included. Substitutions in the amino acid sequence of an antibody chain are described herein by reference to the appropriate reference amino acid sequence, and the same principles as previously described in the context of R100G and A153G substitutions apply to determine position It should be noted that it should. For example, in the foregoing case where the heavy chain may contain one or more amino acid substitutions described, the reference amino acid sequence is EV1018 heavy chain (EV1018; SEQ ID NO: 10). For example, T97A indicates that T (threonine) at position 97 in the EV1018 heavy chain is replaced by A (alanine), and L164A indicates that L (leucine) at position 164 in the EV1018 heavy chain is replaced by A (alanine). Indicates that it will be replaced.
The heavy chain contains one or a combination of two or more of these amino acid substitutions and other amino acid substitutions that do not substantially impair the ability of the antibody or antigen-binding fragment thereof to bind to hGM-CSF and neutralize its activity. Can be included.

Further details regarding embodiments of the present invention are outlined below:
In one embodiment, the heavy chain is one or more of the amino acid substitutions selected from the group consisting of Q3E, T97A, T97V, N95D, N95E, N95K, N95Q, N93Q / N95T, K144R, L164A, and L165A, or 1 Contains one or a combination. In a further embodiment, the heavy chain contains one or more amino acid substitutions selected from the group consisting of Q3E, T97A, T97V, N95D, N95E, N95K, N95Q, N93Q / N95T, K144R, L164A, and L165A. The light chain contains one or both of the following amino acid substitutions R100G and A153G.
Further embodiments include an anti-hGM-CSF monoclonal antibody that binds hGM-CSF or an antigen thereof that binds hGM-CSF, wherein V _H -CDR1 comprises the amino acid residue SYGMH (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333). It is a binding fragment.

Further embodiments include an anti-hGM-CSF monoclonal antibody that binds hGM-CSF or an antigen that binds hGM-CSF, wherein V _H -CDR2 comprises amino acid residues LTYHHGNRKFYADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVKG (SEQ ID NO: 334) It is a binding fragment.
Further embodiments include an anti-hGM-CSF monoclonal antibody that binds hGM-CSF or an antigen thereof that binds hGM-CSF, wherein V _H -CDR3 comprises amino acid residues ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS (SEQ ID NO: 335). It is a binding fragment.

Further embodiments of the invention are as follows:
An anti-hGM-CSF monoclonal antibody that binds hGM-CSF or an antigen-binding fragment thereof that binds hGM-CSF, wherein V _L -CDR1 comprises amino acid residues IGNNNNIGSHAVG (SEQ ID NO: 7) or SGNSSNIGSYAVG (SEQ ID NO: 330).
An anti-hGM-CSF monoclonal antibody that binds hGM-CSF or an antigen-binding fragment thereof that binds hGM-CSF, wherein V _L -CDR2 comprises amino acid residues GRSPPS (SEQ ID NO: 8) or GKSPAS (SEQ ID NO: 331); An anti-hGM-CSF monoclonal antibody that binds hGM-CSF or an antigen-binding fragment thereof that binds hGM-CSF, wherein _L- CDR3 comprises the amino acid residues STWDDSLSAVV (SEQ ID NO: 9) or STWDSLLSAVL (SEQ ID NO: 332).

Additional embodiments of the invention relate to anti-hGM-CSF monoclonal antibodies that bind hGM-CSF or antigen-binding fragments thereof that bind hGM-CSF, further comprising a signal sequence.
The signal sequence includes, but is not limited to, a signal sequence selected from the group consisting of SEQ ID NOs: 324, 325 and 326, allowing or increasing the expression enhancement produced and / or the penetration of the encoded product from the cell. It can be any of a variety of signal sequences known to those of skill in the art, such as any signal sequence that is useful for.
In all embodiments, the anti-hGM-CSF monoclonal antibody may be a human antibody, a humanized antibody, or a single chain antibody.

Antibody Binding Activity Unexpectedly in one aspect, as shown in the examples of this disclosure, the measured binding affinity of an antibody for an antigen (hGM-CSF) is determined by its neutralizing ability (eg, bioactivity of hGM-CSF). There is no linear relationship with the ability to neutralize This observation is contrary to the general concept that high affinity corresponds to high neutralizing ability. For example, an antibody disclosed herein, ie, an antibody that uses a CDR described herein to recognize an epitope described herein, eg, EV1018, is shown in Table 7 as shown in Table 7. It was found that the binding affinity was less than 50% of that of the prior art antibody while showing 100 times higher inhibition than the prior art antibody. This is in contrast to the teaching of WO 2006/122797, which shows that the higher the affinity of the antibody, the higher the activity in the TF-1 assay.

Antibody Neutralizing Activity Human peripheral blood mononuclear cells and tumor cell line TF-1 can proliferate in response to hGM-CSF stimulation. The neutralizing activity of anti-hGM-CSF monoclonal antibodies or antigen-binding fragments thereof is confirmed by measuring their inhibitory effect on proliferation. It is known that human peripheral blood mononuclear cells and TF-1 cells can proliferate when cultured in the presence of hGM-CSF. Such proliferation can be achieved by adding the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof of the present invention to the culture system, or by first adding the anti-hGM-CSF monoclonal antibody or antigen-binding fragment of the present invention and hGM-CSF to the culture system. Can then be inhibited by adding to the culture system. For example, the anti-hGM-CSF monoclonal antibody of the present invention or an antigen-binding fragment thereof has a concentration of about 2 ng / ml (about 14 pM) as neutralizing activity against TF-1 cells whose cell proliferation is induced by hGM-CSF. Compared to negative subjects (31 pg / ml hIgG), it can have a cell growth inhibition rate of 40-60% (about 50%).
The anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof in the present invention has a higher neutralizing ability against hGM-CSF than existing anti-hGM-CSF monoclonal antibodies. Therefore, it is associated with allergic dermatitis, autoimmune disease, chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, interstitial lung disease, rhinitis, arthritis and rheumatoid arthritis, psoriasis, bone marrow It is expected to be applicable as a therapeutic agent used in small amounts or doses for the treatment of various diseases caused by hGM-CSF, such as leukemia leukemia, and multiple sclerosis.

Therapeutic compositions, kits and therapeutic uses The anti-hGM-CSF antibodies and antigen-binding fragments disclosed herein are effective in neutralizing the bioactivity of hGM-CSF, and thus hGM-CSF in a subject. Useful for treating medical conditions (eg, diseases or disorders) associated with (eg, caused by) abnormal expression. Accordingly, such therapeutic uses are intended to be encompassed by the present invention.
The present invention, in certain embodiments, comprises administering to a patient an antibody or antigen-binding fragment thereof or a composition described herein to a patient, as described herein. Provide a method of treating.
As used herein, the term “disease or disorder associated with overexpression of hGM-CSF” shall generally refer to “disease caused by hGM-CSF”. The term is intended to include any disease that causes and exacerbates a disease state when the subject has GM-CSF. The term also includes other diseases that cause and exacerbate the pathophysiology. It is expected that inhibition of GM-CSF bioactivity can alleviate disease symptoms associated with increased GM-CSF and / or alleviate disease progression.

  The terms “disease”, “disorder”, and “condition” are used interchangeably herein. These include respiratory disease, obstructive lung disease of various origins, emphysema of various origins, restrictive lung disease, interstitial lung disease, interstitial lung disease, cystic fibrosis, bronchi of various origins From inflammation, bronchiectasis, ARDS (adult respiratory distress syndrome) and all forms of pulmonary edema, especially COPD (chronic obstructive pulmonary disease), asthma, bronchial asthma, childhood asthma, severe asthma, acute asthma attack and chronic bronchitis By selected obstructive lung disease, COPD (chronic obstructive pulmonary disease) or pulmonary emphysema originating from α1-proteinase inhibitor deficiency, especially allergic alveolitis, asbestosis or silicosis Restricted lung disease induced and restricted lung disease selected from lung tumors such as cancerous lymphangiopathy, bronchoalveolar carcinoma and lymphoma, eg viruses, bacteria Pneumonia caused by infections such as infection by fungi, protozoa, helminths or other pathogens, eg aspiration and left heart failure, radiation pneumonia or fibrosis, eg systemic lupus erythematosus, systemic scleroderma or sarcoidosis, granulomatosis Pneumonia caused by various factors such as collagen disease such as Beck disease, idiopathic interstitial pneumonia or idiopathic pulmonary fibrosis (IPF), etc., bronchitis caused by mucovicidosis, bacterial or viral infection, allergic bronchi Inflammation and toxic bronchitis, bronchiectasis, pulmonary edema, eg toxic pulmonary edema after inhalation or inhalation of toxic and foreign substances, rhinitis, arthritis and related arthropathy, psoriasis, myeloid leukemia, multiple sclerosis, Alzheimer's disease , Nephritis, and chronic atopic dermatitis Is not limited.

As used herein, the terms “treat”, “treatment”, and “treating” refer to the following results: diseases, disorders or conditions associated with aberrant expression and / or activity of human GM-CSF. Obtain all or any of the reduction or improvement of progression, severity, and / or duration, or improvement of one or more of those symptoms resulting from administration of one or more therapeutic agents (eg, GM-CSF antibodies). In general, administration of a therapeutic agent to a subject in an attempt to do so.
As used herein, the term “therapeutically effective amount” refers to abnormal expression and / or activity of human GM-CSF that reduces the severity of a disorder associated with abnormal expression and / or activity of human GM-CSF. Abnormal expression and / or activity of human GM-CSF that ameliorates one or more symptoms of a disorder associated with aberrant expression and / or activity of human GM-CSF that reduces the duration of the disorder involved Disorders that impede the progression of disorders associated with, cause regression of disorders associated with abnormal expression and / or activity of human GM-CSF, or disorders associated with abnormal expression and / or activity of human GM-CSF A therapeutic agent (eg, an antibody that immunospecifically binds to human GM-CSF) that is sufficient to enhance or improve the therapeutic effect (s) of another treatment for It refers to the amount of.

The amount of anti-hGM-CSF monoclonal antibody administered, such as by administering a composition described herein, depends on the condition being treated, the disease or disorder, the age, sex and health status of the subject being treated (individual), and It can vary depending on considerations such as the condition being treated, the severity or degree of progression of the disease or disorder. Appropriate doses can be determined empirically using known methods and referring to the needs of the subject. Typically, the anti-hGM-CSF monoclonal antibody or antigen-binding fragment is administered to the subject at a dose not exceeding 500 mg / dose. Smaller doses can also be administered.
In other embodiments, any of the previously listed anti-GM-CSF antibodies and fragments can be combined. In some embodiments, 2, 3, 4, 5, or more such anti-GM-CSF antibodies, fragments or combinations thereof can be combined.

In some embodiments, any of the previously described embodiments can be further combined with one or more previously known anti-GM-CSF antibodies, fragments, or combinations thereof.
Another embodiment is a therapeutic composition comprising one (one, at least one) anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof described herein. In one embodiment, the therapeutic composition comprises one or more anti-hGM-CSF monoclonal antibodies or antigen-binding fragments that recognize the following ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF, And a pharmaceutically acceptable carrier. The therapeutic composition comprises any of the anti-hGM-CSF monoclonal antibodies or antigen-binding fragments described herein individually (one type or type of anti-hGM-CSF monoclonal antibody or antigen thereof in the therapeutic composition). Binding fragment), or a combination of two or more types or types of anti-hGM-CSF monoclonal antibodies, two or more types or types of antigen-binding fragments thereof, one or more types or types of anti-hGM-CSF monoclonal antibodies And one or more types or types of antigen-binding fragments thereof, one or more types or types of anti-hGM-CSF monoclonal antibodies and two or more types or types of antigen-binding fragments thereof, or two or more types Or types of anti-hGM-CSF monoclonal antibodies and one or more types or types of antigen-binding fragments thereof, It can be included in the suit.

A specific embodiment is a therapeutic composition comprising one (s) anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof that binds hGM-CSF and a pharmaceutically acceptable carrier, This antibody or antigen-binding fragment thereof comprises: (a) (i) a consensus V _H -CDR1-containing sequence FTFSX ₁ X ₂ MH (SEQ ID NO: 314), wherein X ₁ is Y or H and X ₂ is G or A; (Ii) each X _n is independently any natural amino acid, X ₃ is L or V, X ₄ is T or I, X ₅ is Y or W, and X ₆ is R or K A consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), wherein X ₇ is F or Y and X ₈ is R or K; and (iii) each X _n is independently A natural amino acid will, a X ₉ is M or V, comprising a consensus V _H -CDR3 containing sequence X ₁₀ is A or _{G EX n X 9 GX 10 X} n X n DX n ( SEQ ID NO: 316) A heavy chain, or a portion of a heavy chain in the case of an antibody fragment, and (b) (i) a consensus _VL- CDR1-containing sequence in which each X _n is independently any natural amino acid and X ₁₁ is H or Y _{X n GNX n X n NIGSX 11} AVG ( SEQ ID NO: 317), (ii) X ₁₂ is R or K, X ₁₃ is a or P consensus V _L -CDR2 containing sequence GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), and (iii) X ₁₄ is R or S, the light chain comprising a consensus V _L -CDR3 containing sequence STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319) X ₁₅ is V or L or an antibody fragment, If includes the light chain or portion thereof, the anti-hGM-CSF monoclonal antibody or fragment thereof recognizes ELYK in hGM-CSF (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3).

A composition comprising two or more anti-hGM-CSF monoclonal antibodies that bind hGM-CSF, two or more antigen-binding fragments thereof or combinations thereof that bind hGM-CSF, and a pharmaceutically acceptable carrier. The one or more anti-hGM-CSF monoclonal antibodies or one or more antigen-binding fragments thereof may contain discontinuous epitopes ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF. recognize.
Further embodiments described herein include two or more anti-hGM-CSF monoclonal antibodies that bind hGM-CSF, two or more antigen-binding fragments thereof or combinations thereof and a pharmaceutically acceptable carrier. The composition, wherein the one or more antibodies or one or more antigen-binding fragments thereof are (a) (i) a consensus V _H − where X ₁ is Y or H and X ₂ is G or A. CDR1-containing sequence FTFSX ₁ X ₂ MH (SEQ ID NO: 314), (ii) each X _n is independently any natural amino acid, X ₃ is L or V, X ₄ is T or I, X Consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n GX wherein ₅ is Y or W, X ₆ is R or K, X ₇ is F or Y, and X ₈ is R or K _{n X 6 KX 7 YADSVX 8 G} ( Column number 315), and (iii) is any naturally occurring amino acid each X _n is independently, X ₉ is M or V, consensus V _H -CDR3 containing sequence EX _n X X ₁₀ is A or G ₉ GX ₁₀ X _n X _n DX _n (SEQ ID NO: 316) heavy chain, or in the case of antibody fragments, a portion of the heavy chain, and (b) (i) each X _n is independently any natural amino acid , X ₁₁ is H or Y, a consensus V _L -CDR1-containing sequence X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), (ii) X ₁₂ is R or K, and X ₁₃ is A or P A consensus V _L -CDR2 containing sequence GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), and (iii) a consensus V _L -CDR3 containing sequence STWDSX ₁₄ LSAV where X ₁₄ is R or S and X ₁₅ is V or L A light chain comprising X ₁₅ (SEQ ID NO: 319), or in the case of an antibody fragment, a light chain or a portion thereof, the anti-hGM-CSF monoclonal antibody or fragment thereof comprising ELYK (SEQ ID NO: 2) in hGM-CSF and TMMASHYKQH (SEQ ID NO: 3) is recognized.

  Further subject matter described herein is that at least one of the two or more anti-hGM-CSF monoclonal antibodies that bind hGM-CSF is SEQ ID NO: 10-33, 38-80, 152-183, 34-37, 184. A composition comprising a polypeptide selected from the group consisting of ~ 221, 222-245, 320-323 and 348-365. The composition can include other anti-hGM-CSF monoclonal antibodies, such as previously described antibodies, in addition to one or more anti-hGM-CSF monoclonal antibodies described herein. See, for example, PCT Application International Publication No. WO2006 / 122797, PCT Application International Publication No. WO2007 / 092939, and PCT Application International Publication No. WO2006 / 11353.

Kits Accordingly, the anti-hGM-CSF antibodies or antigen-binding fragments thereof disclosed herein and compositions comprising such antibodies can be provided as kits encompassed by the present invention. The kit can be provided in a variety of formats and can include, in addition to the drug (therapeutic composition), a drug or device package and / or label usually derived from a prescription from a physician. The kit generally includes printed instructions for use. In some embodiments, the formulated composition (eg, drug) is provided in one or more containers. In some embodiments, the container may be a device used to dispense medication for administration. For example, the medicament of the present invention can be pretreated (eg, equally divided) into a disposable dispenser and provided as a kit. In some cases, the container is a syringe. The syringe may be filled in advance. In some cases, the disposable container is pre-filled, pre-sealed, and disposable. Depending on the particular drug route of administration and mode of administration, the kit may include appropriate components.

Drug As used herein, a “pharmaceutically acceptable carrier” is any of physiologically compatible solutions, dispersion media, coating agents, antibacterial or antifungal agents, osmotic pressure regulators, and absorption delaying agents. Or include everything. Examples of pharmaceutically acceptable carriers include one or more types of agents such as water, salt solutions, phosphate buffered saline, dextrose, glycerol and ethanol, and combinations thereof. In many cases, it will be preferable to include osmotic agents, such as sugars, polyalcohols, or sodium chloride in the composition. The polyalcohol can include mannitol or sorbitol. In addition, pharmaceutically acceptable carriers can contain minor amounts of auxiliary substances such as wetting agents, emulsifying agents, preservatives, and buffering agents. Auxiliary substances can enhance the storage or effectiveness of the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof in the composition.
Pharmaceutical compositions suitable for parenteral administration can incorporate the anti-hGM-CSF monoclonal antibodies or antigen-binding fragments of the invention herein. Preferably, the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof is for an injection solution containing the antibody in an amount of 0.1 to 250 mg / mL at the time of application of one anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof. Adjust to. On the other hand, when mixing and applying multiple types of antibodies, it is preferable to adjust the antibodies for an injection solution containing the antibodies in an amount of 0.001-10 mg / mL. In addition, multiple types of antibodies can be admixed at will in any ratio.

  Injection solutions formed into liquid or lyophilized formulations can be prepared in flint or amber vials, ampoules, or prefilled syringes. As a buffering agent, L-histidine can be used between pH 15.0 and 7.0 (pH 6.0 is optimal). An L-histidine concentration of 5-10 mM may be optimal. Other agents suitable for buffering agents may be, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride can be applied to the buffer to remove toxicity in a solution at a concentration of 0-300 mM (150 mM is optimal for the dosage form formed in the liquid). The lyophilized dosage form may contain an anti-freezing agent, predominantly sucrose in a proportion of 0-10% (a proportion of 0.5-1.0% may be optimal). Other agents suitable for the cryoprotectant may be trehalose and lactose. The lyophilized dosage form can contain a bulking agent and contains mainly mannitol in a proportion of 1-10% (a proportion of 2-4% may be optimal). As a stabilizer, mainly L-methionine (5-10 mM may be optimal) at a concentration of 1-50 mM can be applied to both liquid or lyophilized dosage forms. Glycine and arginine are included in other suitable bulking agents. Polysorbate 80 may be included as a surfactant in a proportion of 0-0.05% (a proportion of 0.005-0.01% may be optimal). Other surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.

  A variety of dosage forms are applicable to the compositions of the present invention. For example, the composition can have dosage forms formed of liquids, semi-solids, and solids. Solutions (eg, injections or infusions), dispersions, suspensions, tablets, pills, powders, liposomes and suppositories are included. The dosage form preferably depends on the method of administration and the example of treatment. The composition preferably has a dosage form formed of a liquid that is capable of injection and fluid infusion. The composition may be preferred for parenteral administration (eg, can demonstrate intravenous, subcutaneous, abdominal, and intramuscular administration). In a preferred embodiment, the antibody is administered by intravenous infusion or intravenous injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

  The therapeutic composition must generally be produced and stored under sterile and stable conditions. The composition can be formulated into a solution, microemulsion, dispersion, liposome, or other structure suitable to high drug concentration. An injectable sterile solution is prepared by the following procedure. The required amount of active compound (specifically anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof) is mixed in a suitable solvent. If necessary, one or a combination of the aforementioned compounds is mixed with the active compound in a suitable solvent and then sterilized by filtration to prepare a solution. In general, the basic dispersion medium and the active compound are mixed in a sterile medium containing the other required compounds from the aforementioned medium. When preparing sterile injection solutions using sterile lyophilized powder, vacuum drying and spray drying methods are preferably applied as preparation methods. Depending on the method of preparation, any other desired composition is obtained from the active ingredient powder and the sterile solution is subjected to filtration. The fluidity of the solution is properly maintained by the following means. Means are, for example, applying a coating material such as lecithin, maintaining the required particle size in the dispersion, and applying a surfactant. Pharmaceutical absorption delaying agents such as monostearic acid and gelatin are included in the composition, and thus the injectable composition can be absorbed into the human body for an extended period of time.

  The anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof of the present invention can be administered by various methods known in the art. Preferably, administration routes / methods such as subcutaneous injections, intravenous injections, or fluid infusions are applied in various therapies. The route / method of administration depends on the expected results. One skilled in the art can appreciate that implants, transdermal patches and drug delivery systems are included in the route / method of administration. In one embodiment, an antibody such as a controlled release formulation that can control the release of the compound is also applied with the active compound for preparation. The biocompatible polymer is biodegradable. The preparation can include biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Various methods for preparing these dosage forms are allowed as patents and are generally known to those skilled in the art.

  In one embodiment, the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof is orally administered, for example, with an inert diluent or an edible and absorbable carrier. The compound (and other ingredients, if desired) can also be wrapped in hard or soft gelatin capsules, compressed into tablets, or mixed directly into the subject food. During oral administration applied therapeutically, the compound can be mixed with excipients and used in tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. can do. In order to administer the compounds of the present invention by routes other than the parenteral route, it is necessary to coat the compounds with substances to prevent their inactivation and to administer the compounds and substances simultaneously.

Combination therapy Additional active compounds may be supplemented into the composition, and the additional active compounds do not include antibodies to antigens that are not GM-CSF. In one embodiment, the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof of the present invention, along with one or more types of other therapeutic agents useful for treating diseases associated with high amounts of GM-CSF. Formulated or simultaneously administered with other therapeutic agents, where the other therapeutic agent is not an antibody to a protein other than GM-CSF. For example, an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof of the invention is formulated with one or more other anti-GM-CSF antibodies. Combination therapy has the advantage that the therapeutic agent works effectively in small amounts. Any therapeutic agent can avoid the toxicity or complications that can accompany various monotherapy.

As used herein, “administered simultaneously” should be interpreted broadly. For example, when two or more anti-GM-CSF monoclonal antibodies or antigen-binding fragments are administered to a patient, each or some of the antibodies or fragments thereof may be provided as one agent (eg, a composition) It may not be so. In some embodiments, one agent (eg, composition) comprises all of the different types of anti-GM-CSF monoclonal antibodies or antigen-binding fragments thereof that are administered to a subject, so that the antibodies or antigen-binding fragments thereof are in a mixture. Presented to the patient at the same time. In some embodiments, however, two or more agents (eg, compositions) comprising one or more types of antibodies or antigen-binding fragments are administered separately to the subject. When administered “separately”, the agents can be administered, for example, sequentially one after the other. The effects of the first drug and later (second, third, etc.) drug (s) overlap in the time and target cell / tissue in the subject, thereby improving ( As long as it produces a therapeutic result (eg, synergistic), it is to be interpreted as being administered “simultaneously”.
In particular, when more than one anti-hGM-CSF monoclonal antibody of the present invention is applied, it provides cell growth inhibitory activity that is much higher than that provided by a single monoclonal antibody in a pharmaceutical composition (with moderate physiological activity). Can be summed up). Thus, it is possible to provide the therapeutic agent required in that minimum amount.

Therefore, it is a method for improving the activity, comprising two or more types of anti-GM-CSF monoclonal antibodies, or two or more types of anti-hGM-CSF monoclonal antibodies or antigen-binding fragments thereof, and the two or more types of antibodies Are the following (a) and (b):
(A) It has a complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NO: 4 to 9, SEQ ID NO: 330 to 335, SEQ ID NO: 336 to 341, or SEQ ID NO: 342 to 347, and is specific for hGM-CSF Anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof,
(B) the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof of (a), which has an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added, and is specific for hGM-CSF A method of administering a pharmaceutical composition or veterinary pharmaceutical composition characterized in that it is selected from:
Similarly, the present invention relates to a plurality of different types of anti-hGM-CSF monoclonal antibodies of the present invention (hereinafter referred to as “plural types”) or a plurality of types of anti-hGM-CSF antibodies of the present invention. Also included is a method for enhancing activity, characterized in that the pharmaceutical composition or veterinary pharmaceutical composition comprising it is administered simultaneously.

  In any embodiment, the composition can include one type or type of anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof that can neutralize hGM-CSF activity. Alternatively, in any embodiment, the composition can include two or more types or types of anti-hGM-CSF monoclonal antibodies or antigen-binding fragments thereof that can neutralize hGM-CSF activity. Monoclonal antibodies and / or fragments thereof in such compositions may comprise previously disclosed anti-hGM-CSF monoclonal antibodies and / or their additions to one or more antibodies or fragments thereof disclosed herein. Fragments can be included. Anti-hGM-CSF monoclonal antibodies and antigen-binding fragments thereof, and therapeutic compositions, pharmaceutical compositions and pharmaceutical compositions comprising such antibodies and / or antigen-binding fragments thereof, caused by hGM-CSF and A disease, disorder or condition associated with its overexpression (compared to the expression level of hGM-CSF in a control, such as an individual (s) that does not have the disease, condition or disorder) Can be treated or prevented.

A pharmaceutical composition comprising a pharmaceutically acceptable carrier in an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof is considered effective against diseases caused by hGM-CSF. Diseases caused by overproduction of hGM-CSF include
(A) Allergic diseases such as asthma, atopy and hay fever (b) Graft rejection, graft-versus-host disease (GVHD) and (c) autoimmune diseases such as rheumatoid arthritis.

  Without intending to be limited to any particular mechanism, overexpression of GM-CSF is believed to cause, at least in part, many diseases or disorders, such as the diseases or disorders listed herein. However, such clinical conditions may be associated with upregulation of the corresponding receptor (s) or other relevant biological pathway (s) such as mutation of one or more effector pathways. It can also be caused by a failure. Thus, a “disease or disorder associated with overexpression of hGM-CSF” is intended to include conditions that produce results equivalent to overexpression of hGM-CSF.

Production / Preparation In one embodiment, the present invention is an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof disclosed herein for use in medicine.
For the treatment of diseases or disorders associated with overexpression of hGM-CSF (levels caused by GM-CSF, antibodies or antigen-binding fragments thereof can bind to hGM-CSF and neutralize hGM-CSF activity) The use of an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof, such as those described herein, for the manufacture or preparation of a medicament is also an embodiment. Such anti-hGM-CSF monoclonal antibodies and antigen binding fragments thereof may be any of those described herein.

The use of an antibody or antigen-binding fragment thereof that recognizes ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF for the manufacture or preparation of a drug is a further embodiment.
A further embodiment is the use of an anti-hGM-CSF monoclonal antibody and / or antigen-binding fragment thereof for the manufacture or preparation of a medicament, wherein the antibody or fragment thereof is (a) (i) X ₁ is Y or A consensus V _H -CDR1-containing sequence FTFSX ₁ X ₂ MH (SEQ ID NO: 314), (ii) each X _n is independently any natural amino acid, and X ₃ is H and X ₂ is G or A L or V; X ₄ is T or I; X ₅ is Y or W; X ₆ is R or K; X ₇ is F or Y; X ₈ is R or K Consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), and (iii) each X _n is independently any natural amino acid, X ₉ Is M or V and X ₁₀ is A Or a heavy chain comprising the consensus V _H -CDR3-containing sequence EX _n X ₉ GX ₁₀ X _n X _n DX _n (SEQ ID NO: 316) which is G or a portion of the heavy chain in the case of an antibody fragment, and (b) ( i) each X _n is independently any natural amino acid, and X ₁₁ is H or Y, a consensus V _L -CDR1-containing sequence X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), (ii) X Consensus _VL- CDR2-containing sequence GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein ₁₂ is R or K and X ₁₃ is A or P, and (iii) X ₁₄ is R or S, and X ₁₅ is A light chain comprising the consensus V _L -CDR3-containing sequence STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319), which is V or L, or in the case of an antibody fragment, comprises the light chain or a portion thereof.

  In a further aspect, the present invention provides isolated anti-hGM-CSF antibodies or antigen-binding fragments of such antibodies that are useful for the manufacture of a medicament. These agents are useful for the treatment of diseases, conditions or disorders associated with abnormal expression (eg, overexpression) of hGM-CSF. Any of the anti-hGM-CSF monoclonal antibodies and antigen-binding fragments thereof described herein can be used to produce a drug for this purpose.

The antibody or antigen-binding fragment recognizes ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises (a) (i) a consensus V _H -CDR1-containing sequence FTFSX ₁ X ₂ MH (X ₁ is Y or H and X ₂ is G or A SEQ ID NO: 314), is any naturally occurring amino acid independently (ii) each X _n, X ₃ is L or V, a X ₄ is T or I, X ₅ is Y or W, X _A consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G ( ₆ is R or K, X ₇ is F or Y, and X ₈ is R or K SEQ ID NO: 315), and (iii) a consensus V _H -CDR3 containing sequence EX _n X wherein each X _n is independently any natural amino acid, X ₉ is M or V, and X ₁₀ is A or G ₉ weight including _{GX 10 X n X n DX n} ( SEQ ID NO: 316) , Or a portion of the heavy chain in the case of antibody fragments, and (b) (i) is any naturally occurring amino acid each X _n are independently, the consensus V _L -CDR1 containing sequence X _n X ₁₁ is H or Y GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), (ii) Consensus _VL- CDR2-containing sequence GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318) wherein X ₁₂ is R or K and X ₁₃ is A or P And (iii) a light chain comprising the consensus _VL- CDR3-containing sequence STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319), wherein X ₁₄ is R or S and X ₁₅ is V or L, or light for an antibody fragment Includes part of the chain. In one embodiment, the antibody or antigen binding fragment thereof, the hGM-CSF, following 400 pM, (such EV1018 or EV1019) In another embodiment bind with a K _D of less than about 160 pM, K _D is described in Example Decide according to technique.
In other embodiments, any of the previously listed anti-GM-CSF antibodies and fragments can be combined. In some embodiments, 2, 3, 4, 5, or more such anti-GM-CSF antibodies, fragments or combinations thereof can be combined.

Production of Antibodies and Fragments, DNA and Vectors Described below is the production of natural antibodies that recognize the discontinuous epitopes provided in the present invention, but should not be construed as limited to any particular method. The features described can be substituted within the art without departing from the scope of the invention.
The anti-hGM-CSF monoclonal antibody and antigen-binding portion thereof of the present invention include the following steps: isolating cell clones to produce antibodies, selecting antibody-positive cells from the resulting library of antibody-producing cells, And from the blood obtained from patients with idiopathic alveolar proteinosis by purifying antibodies obtained from the supernatant of antibody positive cells by affinity purification.

1) Isolation of fully human antibody-producing cell clones against hGM-CSF From the blood of a patient suffering from idiopathic alveolar proteinosis (IPAP) and having high levels of anti-hGM-CSF monoclonal antibody, B-lymphocytes were obtained. Isolate. B-lymphocytes then induce their proliferation. Methods for inducing its growth are well known. By way of example, a cancer inducer, “Epstein Barr Virus” (hereinafter referred to as EBV) is applied in a transformation method for induced growth (D. Kozbor et al.). More specifically, B-lymphocytes are infected with EBV to induce their proliferation. Proliferated cells are maintained in a library of antibody producing cells.
2) Isolation of monoclonal antibodies from a library of antibody producing cells Monoclonal cells are selected from the induced proliferated cells using well-known methods commonly applied in the production of monoclonal antibodies.
From the library of antibody producing cells, lymphocytes are selected to produce antibodies that bind to hGM-CSF. More specifically, a cell population (clone) producing an antibody that binds to hGM-CSF is selected by limiting dilution. It is preferred to utilize an ELISA that uses a mouse anti-hIgG antibody labeled for detection of hGM-CSF and a fraction that binds to hGM-CSF. A cell population (clone) producing only the desired antibody is obtained by culturing selected antibody-positive cells and repeatedly screening them. The steps up to “isolating an antibody-producing cell clone” are illustrated in the flowchart shown in FIG.
3) Affinity purification using protein A or protein G When purifying anti-hGM-CSF monoclonal antibodies, culturing selected immortalized cells in roller bottles, 2 liter spinner flasks, or other culture systems. Can do. The supernatant is filtered and concentrated. The protein is then purified by affinity chromatography using protein A-Sepharose or protein G-Sepharose or the like (New Jersey, Piscataway, Pharmacia Corp.). After exchanging PBS and buffer solution, the protein concentration is measured by OD at 280 nm, or preferably by turbidimetric analysis. Antibody isotypes are examined by antigen-specific methods for isotype antigens.

The resulting anti-hGM-CSF monoclonal antibody is a fully human antibody produced from B-lymphocytes sensitized in the human body, and thus the anti-hGM-CSF monoclonal antibody does not show much immune response to the antibody. While antibody-producing cells are cloned, B-lymphocytes are infected by EB virus and its growth is induced by the activity of EB virus. Therefore, the antibody-producing cells are cloned by application of the activity of such EB virus. The EB virus method has advantages in producing natural antibodies in the human body and obtaining antibodies with high affinity. For example, the affinity of anti-hGM-CSF monoclonal antibodies is 10-100 times higher than antibodies produced by artificially immunized mice. The library contains a group of B-lymphocytes grown by EB virus infection. Specific antibody-producing cell clones can be isolated from the library to obtain human antibodies.
As noted above, the described features can be substituted or converted within the skill of the art without departing from the scope of the invention. In the present invention, nucleic acids, vectors and host cells can express recombinant antibodies or antigen binding portions thereof. These are also included in the present invention.

  Shown in the table below are EV1018 heavy and light chain variants and EV1019 heavy and light chain variants. These tables give the variants for EV1018 heavy chain and light chain and the variants for EV1019 heavy chain and light chain, respectively, allowing identification of all possible combinations of heavy and light chains for EV1018 and EV1019 respectively. To do. One skilled in the art can generate any combination using methods known in the art and the information provided herein.

  In a further aspect, disclosed herein is a process (method) for producing an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof. Typically, a method for producing an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof obtains or produces DNA consisting essentially of DNA encoding immunoglobulins consisting of heavy and light chains or Fab regions. Inserting the generated DNA into a replicable expression vector in such a way that it is operably linked to an appropriate promoter, thereby generating a vector containing the DNA operably linked to the appropriate promoter Introducing a vector into the host cell, thereby generating a host cell containing the vector, expression of the DNA and generation of the encoding peptide (s) and anti-hGM-CSF monoclonal antibody or antigen-binding fragment Culturing the host cell under conditions suitable for. The resulting anti-hGM-CSF monoclonal antibody or antigen-binding fragment can be recovered from the host cell or host cell culture by methods known in the art.

More specifically, the process includes producing at least V _H -CDR1, V _H -CDR2 and V _H -CDR3, and V _L -CDR1, V _L -CDR2 and V _L -CDR3 in the host cell. . The method comprises introducing a DNA encoding V _H -CDR1, V _H -CDR2, V _H -CDR3, V _L -CDR1, V _L -CDR2 and V _L -CDR3 into a suitable host cell and generating the generated host cell (V _H -CDR1, V _H -CDR2, V _H -CDR3, V _L -CDR1, V _L -CDR2, and V _L -CDR3 host cells containing DNA) expression of DNA (production of coding peptide) And maintaining under conditions suitable for the formation / construction of anti-hGM-CSF monoclonal antibodies or antigen-binding fragments, thereby producing anti-hGM-CSF monoclonal antibodies or antigen-binding fragments. In a specific embodiment, the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof comprises (a) (i) a consensus V _H -CDR1 containing sequence wherein X ₁ is Y or H and X ₂ is G or A. FTFSX ₁ X ₂ MH (SEQ ID NO: 314), (ii) each X _n is independently any natural amino acid, X ₃ is L or V, X ₄ is T or I, and X ₅ is Y Or W, X ₆ is R or K, X ₇ is F or Y, and X ₈ is R or K. Consensus V _H -CDR2-containing sequence X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), and (iii) each X _n is any natural amino acid independently, X ₉ is M or V, consensus V _H X ₁₀ is a or G - CDR3-containing sequence _{EX n X 9 GX 10 X n} X n A X _n heavy chain (SEQ ID NO: 316) or a portion of the heavy chain in the case of antibody fragments, and (b) (i) any naturally occurring amino acid is independently each X _n,, X ₁₁ is H or Y Consensus V _L -CDR1-containing sequence X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), (ii) Consensus V _L -CDR2 containing X ₁₂ is R or K and X ₁₃ is A or P The sequence GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), and (iii) the consensus _VL- CDR3-containing sequence STWDSX ₁₄ LSAX ₁₅ (SEQ ID NO: 319) wherein X ₁₄ is R or S and X ₁₅ is V or L. In the case of an included light chain, or antibody fragment, it includes the light chain or a portion thereof, and the anti-hGM-CSF monoclonal antibody or the resulting antigen-binding fragment binds to hGM-CSF. The DNA encoding V _H -CDR1, V _H -CDR2, V _H -CDR3, V _L -CDR1, V _L -CDR2 and V _L -CDR3 may be one or multiple units (eg, V _H / V _L components can all be encoded by one DNA, and some or all components can be encoded by separate DNA).

  For example, a first DNA can encode a complete heavy chain and a second DNA can encode a complete light chain. The complete heavy chain can be selected from the group consisting of SEQ ID NOs: 10-33, 38-80 and 160-183; 222-245, 320 and 322, and the complete light chain is SEQ ID NOs: 34-37, 202-221. , 321 and 323 can be selected. In one embodiment, the complete heavy chain is selected from the group consisting of SEQ ID NOs: 10-33, 38-80 and 160-183; 222-245, 320 and 322, and the complete light chain is SEQ ID NOs: 34-37, 202. Selected from the group consisting of ˜221, 321 and 323.

  The process can further include isolating the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof.

Further subject matter of the present disclosure is a vector comprising DNA encoding the V _H -CDR1, and / or V _H -CDR2, and / or V _H -CDR3. V _H -CDR1 is SYGMH (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333), V _H -CDR2 is LTYHHGNRKFYADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVKG (SEQ ID NO: 334), and V _H -CDR3 is ESMGAINDN (SEQ ID NO: 334). No. 6) or EWVGGTCDS (SEQ ID NO: 335).

Further subject matter of the present _{specification, V L -CDR1, V L -CDR2} , and V _L -CDR3 a vector comprising DNA encoding, V _L -CDR1 is IGNNNNIGSHAVG (SEQ ID NO: 7) or SGNSSNIGSYAVG (SEQ ID NO: 330 V _L -CDR2 is GRSPPS (SEQ ID NO: 8) or GKSPAS (SEQ ID NO: 331), and V _L -CDR3 is STWDDSLSAVV (SEQ ID NO: 9) or STWDSLSAVL (SEQ ID NO: 332).
For example, heavy and light chain leader sequences are cleaved during the protein maturation process. The cleaved leader sequence has no effect on the final antibody properties. To compensate for the missing sequence, the cloned cDNA is incorporated with synthetic oligonucleotides in a ligation or PCR amplification method. The terms “leader sequence” and “signal sequence” are used interchangeably herein.
In another process, a pair of short overlapping oligonucleotides is used to synthesize a fully variable region, and the resulting oligonucleotide is then amplified in a PCR amplification method so that an artificial clone of the variable region is completely obtained.

  Another aspect of the present invention is an isolated deoxyribonucleic acid (DNA) encoding an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof capable of binding to hGM-CSF and neutralizing the physiological activity of hGM-CSF. The isolated DNA comprises a nucleotide sequence that encodes an amino acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 4-9. The present invention also includes an isolated DNA that encodes an amino acid sequence comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 330-335.

When the isolated deoxyribonucleic acid (DNA) encodes an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof capable of binding to hGM-CSF and neutralizing the physiological activity of hGM-CSF, the following anti-hGM- CSF monoclonal antibodies or antigen-binding fragments thereof are also incorporated in the present invention:
An isolated DNA that can hybridize with the DNA described above under stringent conditions.
The following vectors and host cells are also incorporated in the present invention:
1) A vector incorporating the isolated DNA 2) A host cell incorporating the recombinant expression vector.
Furthermore, by artificially shuffling the V _H and V _L genes as phage fusion proteins using a recently developed phage display method that utilizes genetic engineering techniques to express recombinant antibodies on the phage surface. A specific antibody can also be obtained by expressing the prepared diversified scFv (variable region single chain fragment) antibody.

  The antibody fragment according to the invention can be obtained by any method known to the person skilled in the art, for example by recombinant expression of the fragment encoded by the truncated DNA encoding the antibody, or by proteolysis of the antibody amino acid chain, In this case, one of ordinary skill in the art applies standard biological assays, such as those described herein, to determine which fragments maintained antigen binding function or neutralizing activity.

Specific Antibodies of the Invention The present invention provides the EV1018 antibody itself and numerous variations (eg, variants) of the EV1018 antibody as outlined in Table A. In certain embodiments, the heavy chain comprises one or more amino acid substitutions relative to the wild type heavy chain sequence. In some embodiments, the light chain comprises one or more amino acid substitutions relative to the wild type EV1018 light chain sequence. In some embodiments, the heavy and light chains each comprise one or more amino acid substitutions.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain, EV1018-wt-original (SEQ ID NO: 34), and the following heavy chain variants: EV1018 (SEQ ID NO: 10), EV1018-wt-IgG1KO (sequence No. 11), EV1018-T97A-IgG1KO (SEQ ID NO: 12), EV1018-T97V-IgG1KO (SEQ ID NO: 13), EV1018-N95D-IgG1KO (SEQ ID NO: 14), EV1018-N95E IgG1KO (SEQ ID NO: 15), EV1018-N95K IgG1KO (SEQ ID NO: 16), EV1018-N95Q IgG1KO (SEQ ID NO: 17), EV1018-N93Q-N95T IgG1KO (SEQ ID NO: 18), EV1018-wt IgG1-BI (SEQ ID NO: 19), EV1018-T9 A IgG1-BI (SEQ ID NO: 20), EV1018-T97V IgG1-BI (SEQ ID NO: 21), EV1018-N95D IgG1-BI (SEQ ID NO: 22), EV1018-N95E IgG1-BI (SEQ ID NO: 23), EV1018-N95K IgG1 -BI (SEQ ID NO: 24), EV1018-N95Q IgG1-BI (SEQ ID NO: 25), EV1018-N93Q-N95T IgG1-BI (SEQ ID NO: 26), EV1018-T97A IgG1-original-constant (SEQ ID NO: 27), EV1018- T97V IgG1-original-constant (SEQ ID NO: 28), EV1018-N95D IgG1-original-constant (SEQ ID NO: 29), EV1018-N95E IgG1-original-constant (SEQ ID NO: 30), EV1018-N95K Ig G1-original-constant (SEQ ID NO: 31), EV1018-N95Q IgG1-original-constant (SEQ ID NO: 32), EV1018-N93Q-N95T IgG1-original-constant (SEQ ID NO: 33), EV1018-wt-IgG1-KO-QVQL (SEQ ID NO: 38), EV1018-T97A-IgG1-KO-QVQL (SEQ ID NO: 39), EV1018-T97V-IgG1-KO-QVQL (SEQ ID NO: 40), EV1018-N95D-IgG1-KO-QVQL (SEQ ID NO: 41) EV1018-N95E IgG1-KO-QVQL (SEQ ID NO: 42), EV1018-N95K IgG1-KO-QVQL (SEQ ID NO: 43), EV1018-N95Q IgG1-KO-QVQL (SEQ ID NO: 44), EV1018-N93Q-N9 T IgG1-KO-QVQL (SEQ ID NO: 45), EV1018-wt IgG1-QVQL-BI (SEQ ID NO: 46), EV1018-T97A IgG1-QVQL-BI (SEQ ID NO: 47), EV1018-T97V IgG1-QVQL-BI (sequence) No. 48), EV1018-N95D IgG1-QVQL-BI (SEQ ID NO: 49), EV1018-N95E IgG1-QVQL-BI (SEQ ID NO: 50), EV1018-N95K IgG1-QVQL-BI (SEQ ID NO: 51), EV1018-N95Q IgG1 -QVQL-BI (SEQ ID NO: 52), EV1018-N93Q-N95T IgG1-QVQL-BI (SEQ ID NO: 53), EV1018-IgG2 (SEQ ID NO: 54), EV1018-wt-IgG2 (SEQ ID NO: 55), EV1018 -T97A-IgG2 (SEQ ID NO: 56), EV1018-T97V-IgG2 (SEQ ID NO: 57), EV1018-N95D-IgG2 (SEQ ID NO: 58), EV1018-N95E-IgG2 (SEQ ID NO: 59), EV1018-N95K-IgG2 (sequence) No. 60), EV1018-N95Q-IgG2 (SEQ ID NO: 61), EV1018-N93Q-N95T-IgG2 (SEQ ID NO: 62), EV1018-IgG4 (SEQ ID NO: 63), EV1018-wt-IgG4 (SEQ ID NO: 64), EV1018- T97A-IgG4 (SEQ ID NO: 65), EV1018-T97V-IgG4 (SEQ ID NO: 66), EV1018-N95D-IgG4 (SEQ ID NO: 67), EV1018-N95E-IgG4 (SEQ ID NO: 68), EV1018-N95K-IgG4 SEQ ID NO: 69), EV1018-N95Q-IgG4 (SEQ ID NO: 70), EV1018-N93Q-N95T-IgG4 (SEQ ID NO: 71), EV1018-IgG4-SP (SEQ ID NO: 72), EV1018-wt-IgG4-SP (SEQ ID NO: 71) 73), EV1018-T97A-IgG4-SP (SEQ ID NO: 74), EV1018-T97V-IgG4-SP (SEQ ID NO: 75), EV1018-N95D-IgG4-SP (SEQ ID NO: 76), EV1018-N95E-IgG4-SP ( SEQ ID NO: 77), one of EV1018-N95K-IgG4-SP (SEQ ID NO: 78), EV1018-N95Q-IgG4-SP (SEQ ID NO: 79) and EV1018-N93Q-N95T-IgG4-SP (SEQ ID NO: 80) .

  In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain, EV1018-wt-BI (SEQ ID NO: 35), and the following heavy chain variants: EV1018 (SEQ ID NO: 10), EV1018-wt-IgG1KO (sequence) No. 11), EV1018-T97A-IgG1KO (SEQ ID NO: 12), EV1018-T97V-IgG1KO (SEQ ID NO: 13), EV1018-N95D-IgG1KO (SEQ ID NO: 14), EV1018-N95E IgG1KO (SEQ ID NO: 15), EV1018-N95K IgG1KO (SEQ ID NO: 16), EV1018-N95Q IgG1KO (SEQ ID NO: 17), EV1018-N93Q-N95T IgG1KO (SEQ ID NO: 18), EV1018-wt IgG1-BI (SEQ ID NO: 19), EV1018-T97A IgG1-BI (SEQ ID NO: 20), EV1018-T97V IgG1-BI (SEQ ID NO: 21), EV1018-N95D IgG1-BI (SEQ ID NO: 22), EV1018-N95E IgG1-BI (SEQ ID NO: 23), EV1018-N95K IgG1- BI (SEQ ID NO: 24), EV1018-N95Q IgG1-BI (SEQ ID NO: 25), EV1018-N93Q-N95T IgG1-BI (SEQ ID NO: 26), EV1018-T97A IgG1-original-constant (SEQ ID NO: 27), EV1018-T97V IgG1-original-constant (SEQ ID NO: 28), EV1018-N95D IgG1-original-constant (SEQ ID NO: 29), EV1018-N95E IgG1-original-constant (SEQ ID NO: 30), EV1018-N95K IgG1-original-constant (SEQ ID NO: 31), EV1018-N95Q IgG1-original-constant (SEQ ID NO: 32), EV1018-N93Q-N95T IgG1-original-constant (SEQ ID NO: 33), EV1018-wt-IgG1-KO-QVQL (SEQ ID NO: 38), EV1018-T97A-IgG1-KO-QVQL (SEQ ID NO: 39), EV1018-T97V-IgG1-KO-QVQL (SEQ ID NO: 40), EV1018-N95D-IgG1-KO-QVQL (SEQ ID NO: 41) EV1018-N95E IgG1-KO-QVQL (SEQ ID NO: 42), EV1018-N95K IgG1-KO-QVQL (SEQ ID NO: 43), EV1018-N95Q IgG1-KO-QVQL (SEQ ID NO: 44), EV1018-N93 -N95T IgG1-KO-QVQL (SEQ ID NO: 45), EV1018-wt IgG1-QVQL-BI (SEQ ID NO: 46), EV1018-T97A IgG1-QVQL-BI (SEQ ID NO: 47), EV1018-T97V IgG1-QVQL-BI ( SEQ ID NO: 48), EV1018-N95D IgG1-QVQL-BI (SEQ ID NO: 49), EV1018-N95E IgG1-QVQL-BI (SEQ ID NO: 50), EV1018-N95K IgG1-QVQL-BI (SEQ ID NO: 51), EV1018-N95Q IgG1-QVQL-BI (SEQ ID NO: 52), EV1018-N93Q-N95T IgG1-QVQL-BI (SEQ ID NO: 53), EV1018-IgG2 (SEQ ID NO: 54), EV1018-wt-IgG2 (SEQ ID NO: 55) EV1018-T97A-IgG2 (SEQ ID NO: 56), EV1018-T97V-IgG2 (SEQ ID NO: 57), EV1018-N95D-IgG2 (SEQ ID NO: 58), EV1018-N95E-IgG2 (SEQ ID NO: 59), EV1018-N95K-IgG2 (SEQ ID NO: 60), EV1018-N95Q-IgG2 (SEQ ID NO: 61), EV1018-N93Q-N95T-IgG2 (SEQ ID NO: 62), EV1018-IgG4 (SEQ ID NO: 63), EV1018-wt-IgG4 (SEQ ID NO: 64), EV1018-T97A-IgG4 (SEQ ID NO: 65), EV1018-T97V-IgG4 (SEQ ID NO: 66), EV1018-N95D-IgG4 (SEQ ID NO: 67), EV1018-N95E-IgG4 (SEQ ID NO: 68), EV1018-N9 5K-IgG4 (SEQ ID NO: 69), EV1018-N95Q-IgG4 (SEQ ID NO: 70), EV1018-N93Q-N95T-IgG4 (SEQ ID NO: 71), EV1018-IgG4-SP (SEQ ID NO: 72), EV1018-wt-IgG4- SP (SEQ ID NO: 73), EV1018-T97A-IgG4-SP (SEQ ID NO: 74), EV1018-T97V-IgG4-SP (SEQ ID NO: 75), EV1018-N95D-IgG4-SP (SEQ ID NO: 76), EV1018-N95E- Of IgG4-SP (SEQ ID NO: 77), EV1018-N95K-IgG4-SP (SEQ ID NO: 78), EV1018-N95Q-IgG4-SP (SEQ ID NO: 79) and EV1018-N93Q-N95T-IgG4-SP (SEQ ID NO: 80) Contains one.

  In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain, EV1018-wt-B12 (G1) (SEQ ID NO: 36), and the following heavy chain variants: EV1018 (SEQ ID NO: 10), EV1018-wt- IgG1KO (SEQ ID NO: 11), EV1018-T97A-IgG1KO (SEQ ID NO: 12), EV1018-T97V-IgG1KO (SEQ ID NO: 13), EV1018-N95D-IgG1KO (SEQ ID NO: 14), EV1018-N95E IgG1KO (SEQ ID NO: 15), EV1018-N95K IgG1KO (SEQ ID NO: 16), EV1018-N95Q IgG1KO (SEQ ID NO: 17), EV1018-N93Q-N95T IgG1KO (SEQ ID NO: 18), EV1018-wt IgG1-BI (SEQ ID NO: 19), EV1018 -T97A IgG1-BI (SEQ ID NO: 20), EV1018-T97V IgG1-BI (SEQ ID NO: 21), EV1018-N95D IgG1-BI (SEQ ID NO: 22), EV1018-N95E IgG1-BI (SEQ ID NO: 23), EV1018-N95K IgG1-BI (SEQ ID NO: 24), EV1018-N95Q IgG1-BI (SEQ ID NO: 25), EV1018-N93Q-N95T IgG1-BI (SEQ ID NO: 26), EV1018-T97A IgG1-original-constant (SEQ ID NO: 27), EV1018 -T97V IgG1-original-constant (SEQ ID NO: 28), EV1018-N95D IgG1-original-constant (SEQ ID NO: 29), EV1018-N95E IgG1-original-constant (SEQ ID NO: 30), EV1018 -N95K IgG1-original-constant (SEQ ID NO: 31), EV1018-N95Q IgG1-original-constant (SEQ ID NO: 32), EV1018-N93Q-N95T IgG1-original-constant (SEQ ID NO: 33), EV1018-wt-IgG1-KO -QVQL (SEQ ID NO: 38), EV1018-T97A-IgG1-KO-QVQL (SEQ ID NO: 39), EV1018-T97V-IgG1-KO-QVQL (SEQ ID NO: 40), EV1018-N95D-IgG1-KO-QVQL (SEQ ID NO: 41), EV1018-N95E IgG1-KO-QVQL (SEQ ID NO: 42), EV1018-N95K IgG1-KO-QVQL (SEQ ID NO: 43), EV1018-N95Q IgG1-KO-QVQL (SEQ ID NO: 44), EV101 -N93Q-N95T IgG1-KO-QVQL (SEQ ID NO: 45), EV1018-wt IgG1-QVQL-BI (SEQ ID NO: 46), EV1018-T97A IgG1-QVQL-BI (SEQ ID NO: 47), EV1018-T97V IgG1-QVQL- BI (SEQ ID NO: 48), EV1018-N95D IgG1-QVQL-BI (SEQ ID NO: 49), EV1018-N95E IgG1-QVQL-BI (SEQ ID NO: 50), EV1018-N95K IgG1-QVQL-BI (SEQ ID NO: 51), EV1018 -N95Q IgG1-QVQL-BI (SEQ ID NO: 52), EV1018-N93Q-N95T IgG1-QVQL-BI (SEQ ID NO: 53), EV1018-IgG2 (SEQ ID NO: 54), EV1018-wt-IgG2 (arrangement) No. 55), EV1018-T97A-IgG2 (SEQ ID NO: 56), EV1018-T97V-IgG2 (SEQ ID NO: 57), EV1018-N95D-IgG2 (SEQ ID NO: 58), EV1018-N95E-IgG2 (SEQ ID NO: 59), EV1018- N95K-IgG2 (SEQ ID NO: 60), EV1018-N95Q-IgG2 (SEQ ID NO: 61), EV1018-N93Q-N95T-IgG2 (SEQ ID NO: 62), EV1018-IgG4 (SEQ ID NO: 63), EV1018-wt-IgG4 (SEQ ID NO: 63) 64), EV1018-T97A-IgG4 (SEQ ID NO: 65), EV1018-T97V-IgG4 (SEQ ID NO: 66), EV1018-N95D-IgG4 (SEQ ID NO: 67), EV1018-N95E-IgG4 (SEQ ID NO: 68), EV10 18-N95K-IgG4 (SEQ ID NO: 69), EV1018-N95Q-IgG4 (SEQ ID NO: 70), EV1018-N93Q-N95T-IgG4 (SEQ ID NO: 71), EV1018-IgG4-SP (SEQ ID NO: 72), EV1018-wt- IgG4-SP (SEQ ID NO: 73), EV1018-T97A-IgG4-SP (SEQ ID NO: 74), EV1018-T97V-IgG4-SP (SEQ ID NO: 75), EV1018-N95D-IgG4-SP (SEQ ID NO: 76), EV1018- N95E-IgG4-SP (SEQ ID NO: 77), EV1018-N95K-IgG4-SP (SEQ ID NO: 78), EV1018-N95Q-IgG4-SP (SEQ ID NO: 79) and EV1018-N93Q-N95T-IgG4-SP (SEQ ID NO: 80) Including one of

  In some embodiments, the antibody or antigen-binding fragment thereof is a light chain, EV1018-wt-original-constant (SEQ ID NO: 37), and the following heavy chain variants: EV1018 (SEQ ID NO: 10), EV1018-wt-IgG1KO (SEQ ID NO: 11), EV1018-T97A-IgG1KO (SEQ ID NO: 12), EV1018-T97V-IgG1KO (SEQ ID NO: 13), EV1018-N95D-IgG1KO (SEQ ID NO: 14), EV1018-N95E IgG1KO (SEQ ID NO: 15), EV1018 -N95K IgG1KO (SEQ ID NO: 16), EV1018-N95Q IgG1KO (SEQ ID NO: 17), EV1018-N93Q-N95T IgG1KO (SEQ ID NO: 18), EV1018-wt IgG1-BI (SEQ ID NO: 19), EV101 8-T97A IgG1-BI (SEQ ID NO: 20), EV1018-T97V IgG1-BI (SEQ ID NO: 21), EV1018-N95D IgG1-BI (SEQ ID NO: 22), EV1018-N95E IgG1-BI (SEQ ID NO: 23), EV1018- N95K IgG1-BI (SEQ ID NO: 24), EV1018-N95Q IgG1-BI (SEQ ID NO: 25), EV1018-N93Q-N95T IgG1-BI (SEQ ID NO: 26), EV1018-T97A IgG1-original-constant (SEQ ID NO: 27), EV1018-T97V IgG1-original-constant (SEQ ID NO: 28), EV1018-N95D IgG1-original-constant (SEQ ID NO: 29), EV1018-N95E IgG1-original-constant (SEQ ID NO: 30), EV101 8-N95K IgG1-original-constant (SEQ ID NO: 31), EV1018-N95Q IgG1-original-constant (SEQ ID NO: 32), EV1018-N93Q-N95T IgG1-original-constant (SEQ ID NO: 33), EV1018-wt-IgG1- KO-QVQL (SEQ ID NO: 38), EV1018-T97A-IgG1-KO-QVQL (SEQ ID NO: 39), EV1018-T97V-IgG1-KO-QVQL (SEQ ID NO: 40), EV1018-N95D-IgG1-KO-QVQL (sequence) No. 41), EV1018-N95E IgG1-KO-QVQL (SEQ ID NO: 42), EV1018-N95K IgG1-KO-QVQL (SEQ ID NO: 43), EV1018-N95Q IgG1-KO-QVQL (SEQ ID NO: 44), EV10 8-N93Q-N95T IgG1-KO-QVQL (SEQ ID NO: 45), EV1018-wt IgG1-QVQL-BI (SEQ ID NO: 46), EV1018-T97A IgG1-QVQL-BI (SEQ ID NO: 47), EV1018-T97V IgG1-QVQL -BI (SEQ ID NO: 48), EV1018-N95D IgG1-QVQL-BI (SEQ ID NO: 49), EV1018-N95E IgG1-QVQL-BI (SEQ ID NO: 50), EV1018-N95K IgG1-QVQL-BI (SEQ ID NO: 51), EV1018-N95Q IgG1-QVQL-BI (SEQ ID NO: 52), EV1018-N93Q-N95T IgG1-QVQL-BI (SEQ ID NO: 53), EV1018-IgG2 (SEQ ID NO: 54), EV1018-wt-IgG2 ( Column number 55), EV1018-T97A-IgG2 (SEQ ID NO: 56), EV1018-T97V-IgG2 (SEQ ID NO: 57), EV1018-N95D-IgG2 (SEQ ID NO: 58), EV1018-N95E-IgG2 (SEQ ID NO: 59), EV1018 -N95K-IgG2 (SEQ ID NO: 60), EV1018-N95Q-IgG2 (SEQ ID NO: 61), EV1018-N93Q-N95T-IgG2 (SEQ ID NO: 62), EV1018-IgG4 (SEQ ID NO: 63), EV1018-wt-IgG4 (sequence) No. 64), EV1018-T97A-IgG4 (SEQ ID NO: 65), EV1018-T97V-IgG4 (SEQ ID NO: 66), EV1018-N95D-IgG4 (SEQ ID NO: 67), EV1018-N95E-IgG4 (SEQ ID NO: 68), EV1 018-N95K-IgG4 (SEQ ID NO: 69), EV1018-N95Q-IgG4 (SEQ ID NO: 70), EV1018-N93Q-N95T-IgG4 (SEQ ID NO: 71), EV1018-IgG4-SP (SEQ ID NO: 72), EV1018-wt- IgG4-SP (SEQ ID NO: 73), EV1018-T97A-IgG4-SP (SEQ ID NO: 74), EV1018-T97V-IgG4-SP (SEQ ID NO: 75), EV1018-N95D-IgG4-SP (SEQ ID NO: 76), EV1018- N95E-IgG4-SP (SEQ ID NO: 77), EV1018-N95K-IgG4-SP (SEQ ID NO: 78), EV1018-N95Q-IgG4-SP (SEQ ID NO: 79) and EV1018-N93Q-N95T-IgG4-SP (SEQ ID NO: 80) Including one of .

  The present invention provides the antibody EV1019 itself and a number of variants of the EV1019 antibody, as outlined in Table B. In certain embodiments, the heavy chain comprises one or more amino acid substitutions relative to the wild type heavy chain sequence. In some embodiments, the light chain comprises one or more amino acid substitutions relative to the wild type EV1019 light chain sequence. In some embodiments, the heavy and light chains each comprise one or more amino acid substitutions.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019 (SEQ ID NO: 160), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203). EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (sequence) 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R-BI2 ( SEQ ID NO: 2 3), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (SEQ ID NO: 218) ), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-wt-IgG1-BI (SEQ ID NO: 161), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt- BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207) EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-wt-IgG1KO (SEQ ID NO: 162), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25R BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises heavy chain, EV1019-C105G (SEQ ID NO: 163), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R- BI2 SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (sequence) No. 218), a light chain selected from the group consisting of EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105G-IgG1-BI (SEQ ID NO: 164), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt- BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207) EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- 25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105G-IgG1KO (SEQ ID NO: 165), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105S (SEQ ID NO: 166), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R- BI2 SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (sequence) No. 218), a light chain selected from the group consisting of EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105S-IgG1-BI (SEQ ID NO: 167), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt- BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207) EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- 25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105S-IgG1KO (SEQ ID NO: 168), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105A (SEQ ID NO: 169), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R- BI2 SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (sequence) No. 218), a light chain selected from the group consisting of EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105A-IgG1-BI (SEQ ID NO: 170), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt- BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207) EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- 25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105A-IgG1KO (SEQ ID NO: 171), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105Q (SEQ ID NO: 172), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R- BI2 SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (sequence) No. 218), a light chain selected from the group consisting of EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105Q-IgG1-BI (SEQ ID NO: 173), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt- BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207) EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- 25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105Q-IgG1KO (SEQ ID NO: 174), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises heavy chain, EV1019-C105T (SEQ ID NO: 175), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R- BI2 SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (sequence) No. 218), a light chain selected from the group consisting of EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105T-IgG1-BI (SEQ ID NO: 176), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt- BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207) EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- 25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105T-IgG1KO (SEQ ID NO: 177), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105M (SEQ ID NO: 178), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R- BI2 SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (sequence) No. 218), a light chain selected from the group consisting of EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105M-IgG1-BI (SEQ ID NO: 179), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt- BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207) EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- 25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105M-IgG1KO (SEQ ID NO: 180), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105L (SEQ ID NO: 181), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R- BI2 SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (sequence) No. 218), a light chain selected from the group consisting of EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105L-IgG1-BI (SEQ ID NO: 182), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt- BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207) EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- 25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105L-IgG1KO (SEQ ID NO: 183), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-IgG2 (SEQ ID NO: 222), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-N25R- BI2 ( Column number 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (sequence) No. 218), a light chain selected from the group consisting of EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-wt-IgG4 (SEQ ID NO: 223), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25R-B 2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-wt-IgG4SP (SEQ ID NO: 224), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25R BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises heavy chain, EV1019-C105G-IgG2 (SEQ ID NO: 225), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105G-IgG4 (SEQ ID NO: 226), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105G-IgG4SP (SEQ ID NO: 227), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105S-IgG2 (SEQ ID NO: 228), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105S-IgG4 (SEQ ID NO: 229), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105S-IgG4SP (SEQ ID NO: 230), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105A-IgG2 (SEQ ID NO: 231), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105A-IgG4 (SEQ ID NO: 232), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105A-IgG4SP (SEQ ID NO: 233), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105Q-IgG2 (SEQ ID NO: 234), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105Q-IgG4 (SEQ ID NO: 235), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105Q-IgG4SP (SEQ ID NO: 236), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105T-IgG2 (SEQ ID NO: 237), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105T-IgG4 (SEQ ID NO: 238), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105T-IgG4SP (SEQ ID NO: 239), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105M-IgG2 (SEQ ID NO: 240), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105M-IgG4 (SEQ ID NO: 241), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain, EV1019-C105M-IgG4SP (SEQ ID NO: 242), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105L-IgG2 (SEQ ID NO: 243), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105L-IgG4 (SEQ ID NO: 244), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N25 -BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019- A light chain selected from the group consisting of S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221) is included.

  In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1019-C105L-IgG4SP (SEQ ID NO: 245), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI ( SEQ ID NO: 203), EV1019-wt-BI2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019 -N25G (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019- N 5R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019 A light chain selected from the group consisting of: S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 221).

Furthermore, the variable region of the heavy chain and the variable region of the light chain may be combined to produce a Fab fragment that binds to the antigen. Such combinations that retain the same or equivalent antigen binding affinity are useful for generating various versions of antigen binding fragments, such as single chain variable fragments (scFv). scFv is a fusion of immunoglobulin heavy and light chain variable regions joined together by short (usually serine, glycine) linkers and is well known in the art.
Thus, the variable region of the EV1018 heavy chain may be combined with the variable region of the EV1018 light chain. In some embodiments, the variable region of the heavy and / or light chain may be a variant that includes one or more amino acid changes relative to the wild-type sequence of the chain. Similarly, the variable region of EV1019 heavy chain may be combined with the variable region of EV1019 light chain. In some embodiments, the variable region of the heavy and / or light chain may be a variant that includes one or more amino acid changes relative to the wild-type sequence of the chain.

  In some embodiments, the following 1018 heavy chain variable regions (eg, wild type and variants thereof): EV1018-VH (SEQ ID NO: 348), EV1018-VH-wt (SEQ ID NO: 349), EV1018-VH-T97A ( SEQ ID NO: 350), EV1018-VH-T97V (SEQ ID NO: 351), EV1018-VH-N95D (SEQ ID NO: 352), EV1018-VH-N95E (SEQ ID NO: 353), EV1018-VH-N95K (SEQ ID NO: 354), EV1018 -VH-N95Q (SEQ ID NO: 355), EV1018-VH-N93Q-N95T (SEQ ID NO: 356), EV1018-VH-T97A IgG1-original-constant (SEQ ID NO: 357), EV1018-VH-T97V IgG1-original-constant ( SEQ ID NO: 358), EV1018-VH-N95D IgG1-original-constant (SEQ ID NO: 359), EV1018-VH-N95E IgG1-original-constant (SEQ ID NO: 360), EV1018-VH-N95K IgG1-original-constant (SEQ ID NO: 361), EV1018- Any one of VH-N95Q IgG1-original-constant (SEQ ID NO: 362) and EV1018-VH-N93Q-N95T IgG1-original-constant (SEQ ID NO: 363) was used as a light chain variable region EV1018-VL-wt-original. In combination with (SEQ ID NO: 364), an antigen-binding fragment that binds to hGM-CSF may be formed.

  In some embodiments, the following 1018 heavy chain variable regions (eg, wild type and variants thereof): EV1018-VH (SEQ ID NO: 348), EV1018-VH-wt (SEQ ID NO: 349), EV1018-VH-T97A ( SEQ ID NO: 350), EV1018-VH-T97V (SEQ ID NO: 351), EV1018-VH-N95D (SEQ ID NO: 352), EV1018-VH-N95E (SEQ ID NO: 353), EV1018-VH-N95K (SEQ ID NO: 354), EV1018 -VH-N95Q (SEQ ID NO: 355), EV1018-VH-N93Q-N95T (SEQ ID NO: 356), EV1018-VH-T97A IgG1-original-constant (SEQ ID NO: 357), EV1018-VH-T97V IgG1-original-constant ( SEQ ID NO: 358), EV1018-VH-N95D IgG1-original-constant (SEQ ID NO: 359), EV1018-VH-N95E IgG1-original-constant (SEQ ID NO: 360), EV1018-VH-N95K IgG1-original-constant (SEQ ID NO: 361), EV1018- Any one of VH-N95Q IgG1-original-constant (SEQ ID NO: 362) and EV1018-VH-N93Q-N95T IgG1-original-constant (SEQ ID NO: 363) was selected as the light chain variable region, EV1018-VL-wt- In combination with BI (SEQ ID NO: 365), an antigen-binding fragment that binds to hGM-CSF may be formed.

  Similarly, in some embodiments, the antigen binding fragment of EV1019 comprises the heavy chain variable region, EV1019-VH-wt (SEQ ID NO: 152), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI. (SEQ ID NO: 185), EV1019-VL-N25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187), EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25G (sequence) 189), EV1019-VL-N25T (SEQ ID NO: 190), EV1019-VL-BI-N25T (SEQ ID NO: 191), EV1019-VL-N25R (SEQ ID NO: 192), EV1019-VL-BI-N25R (SEQ ID NO: 193) ), EV1019-VL-N25Q (SEQ ID NO: 1) 4), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198) A light chain variable region selected from the group consisting of: EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201) .

  In some embodiments, the antigen binding fragment of EV1019 comprises the heavy chain variable region, EV1019-VH-C105G (SEQ ID NO: 153), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 184). 185), EV1019-VL-N25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187), EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25G (SEQ ID NO: 189) EV1019-VL-N25T (SEQ ID NO: 190), EV1019-VL-BI-N25T (SEQ ID NO: 191), EV1019-VL-N25R (SEQ ID NO: 192), EV1019-VL-BI-N25R (SEQ ID NO: 193), EV1019 -VL-N25Q (SEQ ID NO: 19 ), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), A light chain variable region selected from the group consisting of EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201) is included.

  In some embodiments, the antigen binding fragment of EV1019 comprises a heavy chain variable region, EV1019-VH-C105S (SEQ ID NO: 154), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 184). 185), EV1019-VL-N25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187), EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25G (SEQ ID NO: 189) EV1019-VL-N25T (SEQ ID NO: 190), EV1019-VL-BI-N25T (SEQ ID NO: 191), EV1019-VL-N25R (SEQ ID NO: 192), EV1019-VL-BI-N25R (SEQ ID NO: 193), EV1019 -VL-N25Q (SEQ ID NO: 19 ), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), A light chain variable region selected from the group consisting of EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201) is included.

  In some embodiments, the antigen binding fragment of EV1019 comprises a heavy chain variable region, EV1019-VH-C105A (SEQ ID NO: 155), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 184). 185), EV1019-VL-N25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187), EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25G (SEQ ID NO: 189) EV1019-VL-N25T (SEQ ID NO: 190), EV1019-VL-BI-N25T (SEQ ID NO: 191), EV1019-VL-N25R (SEQ ID NO: 192), EV1019-VL-BI-N25R (SEQ ID NO: 193), EV1019 -VL-N25Q (SEQ ID NO: 19 ), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), A light chain variable region selected from the group consisting of EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201) is included.

  In some embodiments, the antigen binding fragment of EV1019 comprises a heavy chain variable region, EV1019-VH-C105T (SEQ ID NO: 156), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 184). 185), EV1019-VL-N25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187), EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25G (SEQ ID NO: 189) EV1019-VL-N25T (SEQ ID NO: 190), EV1019-VL-BI-N25T (SEQ ID NO: 191), EV1019-VL-N25R (SEQ ID NO: 192), EV1019-VL-BI-N25R (SEQ ID NO: 193), EV1019 -VL-N25Q (SEQ ID NO: 19 ), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), A light chain variable region selected from the group consisting of EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201) is included.

  In some embodiments, the antigen binding fragment of EV1019 comprises a heavy chain variable region, EV1019-VH-C105M (SEQ ID NO: 157), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 184). 185), EV1019-VL-N25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187), EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25G (SEQ ID NO: 189) EV1019-VL-N25T (SEQ ID NO: 190), EV1019-VL-BI-N25T (SEQ ID NO: 191), EV1019-VL-N25R (SEQ ID NO: 192), EV1019-VL-BI-N25R (SEQ ID NO: 193), EV1019 -VL-N25Q (SEQ ID NO: 19 ), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), A light chain variable region selected from the group consisting of EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201) is included.

  In some embodiments, the antigen binding fragment of EV1019 comprises a heavy chain variable region, EV1019-VH-C105Q (SEQ ID NO: 158), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 184). 185), EV1019-VL-N25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187), EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25G (SEQ ID NO: 189) EV1019-VL-N25T (SEQ ID NO: 190), EV1019-VL-BI-N25T (SEQ ID NO: 191), EV1019-VL-N25R (SEQ ID NO: 192), EV1019-VL-BI-N25R (SEQ ID NO: 193), EV1019 -VL-N25Q (SEQ ID NO: 19 ), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), A light chain variable region selected from the group consisting of EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201) is included.

  In some embodiments, the antigen binding fragment of EV1019 comprises the heavy chain variable region, EV1019-VH-C105L (SEQ ID NO: 159), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 184). 185), EV1019-VL-N25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187), EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25G (SEQ ID NO: 189) EV1019-VL-N25T (SEQ ID NO: 190), EV1019-VL-BI-N25T (SEQ ID NO: 191), EV1019-VL-N25R (SEQ ID NO: 192), EV1019-VL-BI-N25R (SEQ ID NO: 193), EV1019 -VL-N25Q (SEQ ID NO: 19 ), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), A light chain variable region selected from the group consisting of EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201) is included.

Variations within the present invention The present invention includes when two or more types of full length anti-hGM-CSF antibody or antigen-binding portion thereof, or anti-hGM-CSF antibody portion are administered simultaneously. Accordingly, bispecific antibodies and multispecific antibodies that recognize hGM-CSF are also included in the present invention.
While variations of the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof have been described in the above description, the described features may be replaced or converted in the art without departing from the scope of the invention .

That is, the following antibodies or antigen-binding portions thereof are included in the scope of the present invention:
(I) Full length antibody and antigen binding portion thereof (II) Antibody portion, and (III) Recombinant human monoclonal antibody, monoclonal antibody (including chimeric and humanized antibodies), or specific for hGM-CSF Its antigen-binding portion capable of binding to and neutralizing its bioactivity (recombinant human monoclonal antibodies represent variable regions and CDRs, SEQ ID NOs: 4-9 and 330-335, SEQ ID NO: 364) And 365, and any well known technique based on the amino acid sequences of SEQ ID NOs: 348-363, SEQ ID NOs: 184-201, and SEQ ID NOs: 152-159).
The specific antibody or antigen-binding portion thereof generated by the above method is applied based on at least one amino acid sequence selected from each group consisting of SEQ ID NOs: 4-9 or 330-335 described herein If so, antibodies or antigen-binding portions thereof are also incorporated in the present invention.

List of Antibody Variants The table provided below provides a list of useful antibody chains and fragments for the EV1018 and EV1019 antibodies, respectively.
Table A: List of heavy and light chain variants and variable regions for EV1018 antibody

Table B: List of heavy and light chain variants and variable regions for EV1019 antibody

Various embodiments of the invention
In the following embodiments, the present invention will be described in more detail. The description of the embodiments is constructed in a manner known from the claims making point. This is because it appears to be useful to illustrate in the specification the teachings that can be derived from the scope of protection conferred by this patent application.
Item 1. One embodiment is an isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof that recognizes ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF (SEQ ID NO: 1).
Item 2. Another embodiment is:
(A) Consensus V _H -CDR1-containing sequences, consensus V _H CDR2-containing sequences and consensus V _H A heavy chain comprising a CDR3-containing sequence,
(I) Consensus V _H -CDR1 containing sequence is FTFSX ₁ X ₂ MH (SEQ ID NO: 314), wherein X ₁ Is Y or H and X ₂ Is G or A,
(Ii) Consensus V _H The CDR2-containing sequence is X _Three X _Four X _Five HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), wherein each X _n Are independently any natural amino acid and X _Three Is L or V and X _Four Is T or I and X _Five Is Y or W and X ₆ Is R or K and X ₇ Is F or Y and X ₈ Is R or K;
(Iii) Consensus V _H -CDR3 containing sequence is EX _n X ₉ GX _Ten X _n X _n DX _n (SEQ ID NO: 316), wherein each X _n Are independently any natural amino acid and X ₉ Is M or V and X _Ten A heavy chain wherein A or G; and
(B) Consensus V _L -CDR1-containing sequences, consensus V _L CDR2-containing sequences and consensus V _L A light chain comprising a CDR3-containing sequence,
(I) Consensus V _L The CDR1-containing sequence is X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), wherein each X _n Are independently any natural amino acid and X ₁₁ Is H or Y,
(Ii) Consensus V _L -CDR2 containing sequence is GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein X ₁₂ Is R or K and X ₁₃ Is A or P,
(Iii) Consensus V _L -CDR3 containing sequence is STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319), wherein X ₁₄ Is R or S and X ₁₅ Light chain in which is V or L
An isolated anti-hGM-CSF monoclonal antibody or an antigen-binding fragment thereof, characterized in that In other embodiments, the antibody or antigen-binding fragment of item 1 or item 2 binds to hGM-CSF, preferably with a K of less than 450 pM. _D Bind specifically.
Item 3. In another embodiment, in addition to the properties listed in item 1 or 2, human GM-CSF has a K of less than 400 pM. _D Or an antigen-binding fragment thereof disclosed in item 1 or 2.
Item 4. Another embodiment is K _D The antibody or antigen-binding fragment thereof disclosed in item 3, wherein is less than 160 pM.
Item 5. Another embodiment neutralizes hGM-CSF activity and, therefore, the IC50 value of the antibody or antigen-binding fragment thereof is less than 100 pM as measured by ED80 in a TF-1 proliferation assay, any of items 1-4 The antibody or antigen-binding fragment thereof disclosed in item 1.
Item 6. Another embodiment is:
6. The antibody or antigen-binding fragment thereof disclosed in item 5, having an IC50 value of less than 40 pM or less than 30 pM or less than 25 pM. Further preferred antibodies of the invention or antigen binding fragments thereof have an IC50 value of less than 20 pM, less than 25 pM, less than 30 pM or less than 40 pM as measured by ED80 in a TF-1 proliferation assay.
Item 7. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in item 5 having an IC50 value of less than 20 pM.
Item 8. In another embodiment, the heavy chain is gamma 1 (γ ₁ ), Gamma 2 (γ ₂ ), Gamma 3 (γ _Three ) And gamma 4 (γ _Four The antibody or antigen-binding fragment thereof disclosed in any one of items 1 to 7, selected from the group consisting of:
Item 9. Another embodiment is disclosed in any of items 1-8, wherein the heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-33, 38-80, 160-183, 222-244 and 245. Antibody or antigen-binding fragment thereof.
Item 10. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in any of items 1-9, wherein the light chain is a lambda light chain.
Item 11. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in item 10, wherein the lambda light chain comprises at least one or both of the following amino acid substitutions: R100G or A153G.
Item 12. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in any of items 1-11, wherein the light chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-37, 202-220 and 221. is there.
Item 13. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in any of items 1-9, wherein the light chain is a kappa light chain.
Item 14. In another embodiment, the heavy chain is gamma 1 (γ ₁ ), Gamma 2 (γ ₂ ), Gamma 3 (γ _Three ) And gamma 4 (γ _Four The antibody or antigen-binding fragment thereof disclosed in any one of items 1 to 13, wherein the light chain is a lambda light chain.
Item 15. Another embodiment is one in which the heavy chain comprises one or more amino acid substitutions selected from the group consisting of Q3E, T97A, T97V, N95D, N95E, N95K, N95Q, N93Q / N95T, K144R, L164A and L165A. The antibody or antigen-binding fragment thereof disclosed in any one of items 1 to 13.
Item 16. Another embodiment is V _H -CDR1 is an antibody or antigen-binding fragment thereof disclosed in any of items 1-15, wherein the CDR1 comprises the amino acid sequence SYGMH (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333).
Item 17. Another embodiment is V _H -CDR2 is the antibody or antigen-binding fragment thereof disclosed in any of items 1-16, wherein CDR2 comprises the amino acid sequence LTYHHGNRKFYADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVKG (SEQ ID NO: 334).
Item 18. Another embodiment is V _H -The antibody or antigen-binding fragment thereof disclosed in any of items 1-17, wherein CDR3 comprises the amino acid sequence ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS (SEQ ID NO: 335).
Item 19. Another embodiment is V _L -CDR1 is an antibody or antigen-binding fragment thereof disclosed in any of items 1-18, wherein CDR1 comprises the amino acid sequence IGNNNNIGSHAVG (SEQ ID NO: 7) or SGNSSNIGSHAVAVG (SEQ ID NO: 330).
Item 20. Another embodiment is V _L -CDR2 is the antibody or antigen-binding fragment thereof disclosed in any of items 1-19, wherein the CDR2 comprises the amino acid sequence GRSPPS (SEQ ID NO: 8) or GKSPAS (SEQ ID NO: 331).
Item 21. Another embodiment is V _L -CDR3 is an antibody or antigen-binding fragment thereof disclosed in any of items 1-20, wherein CDR3 comprises amino acid residues STWDDSLSAVV (SEQ ID NO: 9) or STWDDSLSAVL (SEQ ID NO: 332).
Item 22. Another embodiment is an isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof comprising 6 different CDRs, wherein the sequences of the 6 CDRs are SEQ ID NOs: 4-9. In other embodiments, the antibody or antigen-binding fragment of item 22 has a hGM-CSF with a K of preferably less than 450 pM, preferably 400 pM or 160 pM. _D Bind specifically.
Item 22A. Another embodiment is an isolated anti-hGM-CSF monoclonal antibody disclosed in item 1 in combination with an isolated anti-hGM-CSF monoclonal antibody disclosed in any of items 2-22 or an antigen-binding fragment thereof. Or an antigen-binding fragment thereof.
Item 23. Another embodiment is: (i) Gamma 1 (γ ₁ ), Gamma 2 (γ ₂ ), Gamma 3 (γ _Three ) And gamma 4 (γ _Four The antibody or antigen-binding fragment thereof disclosed in item 22, comprising a heavy chain selected from the group consisting of:) and a fragment thereof; and (ii) a light chain that is a kappa light chain.
Item 24. Another embodiment is: (i) Gamma 1 (γ ₁ ), Gamma 2 (γ ₂ ), Gamma 3 (γ _Three ) And gamma 4 (γ _Four The antibody or antigen-binding fragment thereof disclosed in item 22, comprising a heavy chain selected from the group consisting of:) and a fragment thereof, and (ii) a light chain that is a lambda light chain.
Item 25. Another embodiment comprises an isolated anti-hGM-CSF monoclonal antibody that specifically binds hGM-CSF, comprising six different CDRs, wherein the sequences of the six CDRs are SEQ ID NOs: 330-335 or An antigen-binding fragment thereof.
Item 26. Another embodiment is: (i) Gamma 1 (γ ₁ ), Gamma 2 (γ ₂ ), Gamma 3 (γ _Three ) And gamma 4 (γ _Four 26) The antibody or antigen-binding fragment thereof disclosed in item 25, comprising a heavy chain selected from the group consisting of or a fragment thereof, and (ii) a light chain that is a kappa light chain.
Item 27. Another embodiment is: (i) Gamma 1 (γ ₁ ), Gamma 2 (γ ₂ ), Gamma 3 (γ _Three ) And gamma 4 (γ _Four The antibody or antigen-binding fragment thereof disclosed in item 25, comprising a heavy chain selected from the group consisting of: and a light chain that is (ii) a lambda light chain.
Item 28. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in any of items 1-27, further comprising a signal sequence.
Item 29. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in item 28, wherein the signal sequence is selected from the group consisting of SEQ ID NOs: 324, 325 and 326.
Item 30. Another embodiment comprises a heavy chain having a heavy chain sequence and a light chain having a light chain sequence, wherein the heavy chain sequence is selected from the group consisting of SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-79 and 80 An isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof, wherein the light chain sequence is SEQ ID NO: 34.
Item 31. Another embodiment comprises a heavy chain having a heavy chain sequence and a light chain having a light chain sequence, wherein the heavy chain sequence is selected from the group consisting of SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-79 and 80 An isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof, wherein the light chain sequence is SEQ ID NO: 35.
Item 32. Another embodiment comprises a heavy chain having a heavy chain sequence and a light chain having a light chain sequence, wherein the heavy chain sequence is selected from the group consisting of SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-79 and 80 An isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof, wherein the light chain sequence is SEQ ID NO: 36.
Item 33. Another embodiment comprises a heavy chain having a heavy chain sequence and a light chain having a light chain sequence, wherein the heavy chain sequence is selected from the group consisting of SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-79 and 80 An isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof, wherein the light chain sequence is SEQ ID NO: 37.
Item 34. Another embodiment comprises a heavy chain having a heavy chain sequence and a light chain having a light chain sequence, wherein said heavy chain sequence is selected from the group consisting of SEQ ID NO: 160 or SEQ ID NOs: 161-244 and 245 An isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof, wherein the light chain sequence is SEQ ID NO: 202 or a variant thereof selected from the group consisting of SEQ ID NOs: 203-220 and 221.
Item 35. Another embodiment is the amino acid sequence of the heavy chain variable region, wherein the heavy chain variable region is an amino acid sequence thereof selected from the group consisting of SEQ ID NO: 152 or SEQ ID NOs: 153 to 158 and 159, and the light chain variable region Is an isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof comprising said light chain variable region which is a variant selected from the group consisting of SEQ ID NO: 184 or SEQ ID NOs: 185-200 and 201.
Item 36. Another embodiment is the antibody disclosed in item 35 or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 152.
Item 37. Another embodiment is the antibody disclosed in item 35 or an antigen-binding fragment thereof, wherein the light chain variable region amino acid sequence is SEQ ID NO: 184.
Item 38. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in item 35, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 152 and the amino acid sequence of the light chain variable region is 184.
Item 39. In another embodiment, the antibody is an IgG ₁ (Λ) The antibody or antigen-binding fragment thereof disclosed in any one of items 35 to 38 belonging to a class (subclass).
Item 40. Another embodiment is the heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is a variant thereof selected from the group consisting of SEQ ID NO: 348 or SEQ ID NOs: 349-362 and 363, and the amino acid sequence of the light chain variable region Is an isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof comprising the light chain variable region wherein SEQ ID NO: 364 or SEQ ID NO: 365.
Item 41. Another embodiment is the antibody disclosed in item 40 or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 348.
Item 42. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in item 40, wherein the light chain variable region amino acid sequence is SEQ ID NO: 364.
Item 43. Another embodiment is the antibody disclosed in item 40 or an antigen-binding fragment thereof, wherein the light chain variable region amino acid sequence is SEQ ID NO: 365.
Item 44. In another embodiment, the antibody is an IgG ₁ (Λ) The antibody or antigen-binding fragment thereof disclosed in any one of items 40 to 43, belonging to a class (subclass).
Item 45. Another embodiment is an isolated nucleic acid encoding the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof disclosed in items 1-44.
Item 46. Another embodiment is the nucleic acid disclosed in item 45, wherein the nucleic acid is DNA.
Item 47. Another embodiment is a vector comprising the DNA disclosed in item 46.
Item 48. Another embodiment is a host cell comprising the vector disclosed in item 47, wherein the vector is an expression vector.
Item 49. Another embodiment is a kit comprising (a) the antibody or antigen-binding fragment thereof disclosed in any of items 1-44, and (b) one or more containers containing the antibody or antigen-binding fragment thereof. .
Item 50. Another embodiment is the antibody or antigen-binding fragment thereof disclosed in any of items 1-44 for use in medicine.
Item 51. Another embodiment is a composition comprising the antibody or antigen-binding fragment thereof disclosed in any of items 1-44 and a pharmaceutically acceptable carrier.
Item 52. Another embodiment is such that the composition comprises a plurality of said antibodies, a plurality of said antigen binding fragments, or at least one said antibody and at least one said antigen binding fragment, all of which bind to hGM-CSF. 52. The composition disclosed in item 51, further comprising a second isolated antibody or antigen-binding fragment thereof that binds to hGM-CSF.
Item 53. Another embodiment is a polypeptide wherein at least one of said antibodies or antigen-binding fragments thereof has a sequence selected from the group consisting of SEQ ID NOs: 10-80, 152-245, 320-323, 348-364 and 365 52. The composition of item 52, wherein
Item 54. Another embodiment is a kit comprising the composition disclosed in any of items 51-53 and one or more containers containing said composition.
Item 55. Another embodiment is:
56. The kit disclosed in item 49 or item 54, further comprising instructions.
Item 56. Another embodiment provides an antibody disclosed in any of items 1-44 and 51-53 for manufacturing or preparing a medicament for treating a disease or disorder associated with overexpression of hGM-CSF in a subject, or An antigen-binding fragment or composition thereof, which is an antibody or antigen-binding fragment or composition thereof capable of binding to hGM-CSF and neutralizing hGM-CSF activity.
Item 57. In another embodiment, the disease or disorder is chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, interstitial lung disease, rhinitis, arthritis and related joint diseases, psoriasis, myeloid leukemia and multiple Use as disclosed in item 56, selected from the group consisting of sclerosis.
Item 58. Another embodiment is the use disclosed in item 56 or 57, wherein the antibody or antigen-binding fragment is administered to the subject at a dose not exceeding 500 mg.
Item 59. Another embodiment of the antibody or antigen-binding fragment or composition thereof disclosed in any of items 1-44 and 51-53 for treating a disease or disorder associated with overexpression of hGM-CSF in a subject Use wherein the antibody or antigen-binding fragment thereof can bind to hGM-CSF and neutralize hGM-CSF activity.
Item 60. In another embodiment, the disease or disorder is chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, interstitial lung disease, rhinitis, arthritis and related joint diseases, psoriasis, myeloid leukemia and multiple The use disclosed in item 59, selected from the group consisting of sclerosis.
Item 61. Another embodiment is the use disclosed in item 59 or 60, wherein the antibody or antigen-binding fragment is administered to the subject at a dose not exceeding 500 mg.
Item 62. Another embodiment is an epitope of hGM-CSF in a polypeptide disclosed in SEQ ID NOs: 2 and 3, wherein said epitope is recognized by an antibody or antigen-binding fragment thereof disclosed in item 22 or 25, wherein said polypeptide sequence Is an epitope representing a discontinuous portion of hGM-CSF (SEQ ID NO: 1).
Item 63. Another embodiment is an epitope that is a discontinuous portion of human GM-CSF, comprising amino acid residues 77-80 of human GM-CSF (SEQ ID NO: 1) and amino acids of human GM-CSF (SEQ ID NO: 1) An epitope recognized by an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof comprising residues 95-104 and comprising six different CDRs disclosed in SEQ ID NOs: 4-9 or SEQ ID NOs: 330-335.
Item 64. Another embodiment is a method of producing in a host cell an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof that binds hGM-CSF, wherein said antibody or antigen-binding fragment thereof is at least consensus V. _H -CDR1-containing sequences, consensus V _H -CDR2 containing sequence, consensus V _H -CDR3-containing sequences, consensus V _L -CDR1-containing sequences, consensus V _L CDR2-containing sequences and consensus V _L -Comprising a CDR3-containing sequence, the method comprising:
(I) at least the consensus V _H -CDR1-containing sequences, the consensus V _H -CDR2 containing sequence, said consensus V _H -CDR3-containing sequences, said consensus V _L -CDR1-containing sequences, the consensus V _L -CDR2 containing sequences and said consensus V _L Obtaining a host cell comprising at least one DNA sequence encoding a CDR3-containing sequence,
(A) Consensus V _H -CDR1 containing sequence is FTFSX ₁ X ₂ MH (SEQ ID NO: 314), wherein X ₁ Is Y or H and X ₂ Is G or A,
(B) Consensus V _H The CDR2-containing sequence is X _Three X _Four X _Five HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), wherein each X _n Are independently any natural amino acid and X _Three Is L or V and X _Four Is T or I and X _Five Is Y or W and X ₆ Is R or K and X ₇ Is F or Y and X ₈ Is R or K;
(C) Consensus V _H -CDR3 containing sequence is EX _n X ₉ GX _Ten X _n X _n DX _n (SEQ ID NO: 316), wherein each X _n Are independently any natural amino acid and X ₉ Is M or V and X _Ten Is A or G,
(D) Consensus V _L The CDR1-containing sequence is X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), wherein each X _n Are independently any natural amino acid and X ₁₁ Is H or Y,
(E) Consensus V _L -CDR2 containing sequence is GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein X ₁₂ Is R or K and X ₁₃ Is A or P,
(F) Consensus V _L -CDR3 containing sequence is STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319), wherein X ₁₄ Is R or S and X ₁₅ Is V or L, and
(Ii) culturing host cells under conditions suitable for expression of DNA and production of antibodies or antigen-binding fragments thereof.
It is a method including.
Item 65. In another embodiment, the at least one DNA encodes a heavy chain or portion thereof and a light chain or portion thereof, wherein the heavy chain or portion thereof is SEQ ID NO: 10-33, 38-80, 152-183, 222. Having a sequence selected from the group consisting of ~ 245, 348-362 and 363, wherein the light chain or part thereof has a sequence selected from the group consisting of SEQ ID NOs: 34-37, 184-221, 364 and 365 , The method disclosed in item 64.
Item 66. Another embodiment is the method disclosed in item 64 or 65, further comprising isolating the antibody or antigen-binding fragment thereof.
Item 67. Another embodiment is the method disclosed in any of items 64-66, further comprising preparing a composition comprising an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
Item 68. Another embodiment is V _H -CDR1, V _H -CDR2 and V _H A vector comprising a DNA sequence encoding CDR3,
V _H -CDR1 is SYGMH (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333);
V _H -CDR2 is LTYHHGNRKFYADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVKG (SEQ ID NO: 334);
V _H -A vector in which CDR3 is ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS (SEQ ID NO: 335).
Item 69. Another embodiment is V _L -CDR1, V _L -CDR2 and V _L A vector comprising a DNA sequence encoding CDR3,
V _L -CDR1 is IGNNNNIGSHAVG (SEQ ID NO: 7) or SGNSSNIGSYAVG (SEQ ID NO: 330);
V _L -CDR2 is GRPSPPSG (SEQ ID NO: 8) or GKSPASG (SEQ ID NO: 331);
V _L -A vector in which CDR3 is STWDDSLSAVV (SEQ ID NO: 9) or STWDDSLSAVL (SEQ ID NO: 332).
Item 70. Another embodiment is:
(I) contacting a biological sample or peptide library with a probe comprising an epitope;
(Ii) isolating a molecule that specifically binds to the probe; and
(Iii) identifying the molecule
A method for identifying a molecule that binds to the epitope disclosed in item 62 or 63.
Item 71. Another embodiment is:
(Iv) producing the molecule of (iii)
The method disclosed in item 70, further comprising:
Item 72. Another embodiment is the method disclosed in item 70 or 71, wherein the molecule is an anti-hGM-CSF monoclonal antibody or an antigen-binding fragment thereof.
Item 73. Another embodiment is the method disclosed in any of items 70-72, wherein the probe further comprises a detectable marker.
Item 74. Another embodiment is the method disclosed in any one of items 70-73, wherein the probe is immobilized.
Item 75. Another embodiment is an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof that binds to hGM-CSF (hGM-CSF) and can neutralize the bioactivity of hGM-CSF (hGM-CSF). An anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof, comprising a complementarity determining region (CDR) represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4 to 9 It is.
Item 76. Another embodiment is an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof that binds to hGM-CSF and can neutralize the bioactivity of hGM-CSF, from the group consisting of SEQ ID NOs: 330-335 An anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof, characterized in that it has a complementarity determining region (CDR) represented by one or more selected amino acid sequences.
Item 77. Another embodiment disclosed in item 75 or item 76, characterized in that it has an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted or added within a complementarity determining region (CDR). Anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof.
Item 78. Another embodiment is according to item 75 to item 77, characterized in that when TF-1 cells proliferate upon induction of hGM-CSF, the concentration of TF-1 cells is inhibited by about 50% at a concentration of about 14 pM. The disclosed anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof.
Item 79. Another embodiment is the anti-hGM-CSF monoclonal antibody disclosed in item 78 or an antigen-binding portion thereof, characterized in that it inhibits proliferation of peripheral blood dendritic cells.
Item 80. Another embodiment is 4 × 10 4 for hGM-CSF. ^-Ten K below M _D The anti-hGM-CSF monoclonal antibody disclosed in item 78 or an antigen-binding portion thereof, characterized by having a high affinity for the value.
Item 81. Another embodiment is IgG ₁ (Λ) The anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof disclosed in items 75 to 80, which belongs to the class (subclass).
Item 82. Another embodiment is the anti-hGM-CSF monoclonal antibody disclosed in items 75 to 81 or an antigen-binding portion thereof, characterized in that it is a human monoclonal antibody.
Item 83. Another embodiment is a pharmaceutical composition for a disease caused by hGM-CSF comprising an anti-hGM-CSF monoclonal antibody or antigen-binding moiety disclosed in items 75-82 and a pharmaceutically acceptable carrier. .
Item 84. In another embodiment, the disease caused by overproduction of hGM-CSF is
(A) allergic diseases such as asthma, atopy and hay fever,
(B) graft rejection, graft versus host disease (GVHD), and
(C) Autoimmune diseases such as rheumatoid arthritis
84. The pharmaceutical composition disclosed in item 83, wherein the pharmaceutical composition is any one selected from the group consisting of:
Item 85. Another embodiment is an isolated deoxyribonucleic acid (DNA) encoding an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof that can bind to hGM-CSF and neutralize the bioactivity of hGM-CSF. An isolated DNA, characterized in that it encodes an amino acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 4 to 9.
Item 86. Another embodiment is an isolated DNA encoding an anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof that binds to hGM-CSF and is capable of neutralizing the bioactivity of hGM-CSF, comprising: An isolated DNA characterized in that it encodes an amino acid sequence comprising at least one selected from the group consisting of numbers 330 to 335.
Item 87. Another embodiment is an isolated DNA that can hybridize under stringent conditions with the DNA disclosed in item 85 or item 86.
Item 88. Another embodiment is a vector in which the isolated DNA disclosed in any of items 85 to 87 is incorporated.
Item 89. Another embodiment is a host cell into which the recombinant expression vector disclosed in item 88 has been introduced.
Item 90. Another embodiment is a method for enhancing activity, characterized in that multiple types of anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof that are specific for the same specific antigen are administered simultaneously. Is the method.
Item 91. In another embodiment, the plurality of types of anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof are the following (a) and (b):
(A) It has a complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NO: 4 to 9, SEQ ID NO: 330 to 335, SEQ ID NO: 336 to 341, or SEQ ID NO: 342 to 347, and is specific for hGM-CSF An anti-hGM-CSF monoclonal antibody or an antigen-binding portion thereof,
(B) the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof of (a) having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added, and specific for hGM-CSF
A method for enhancing the activity disclosed in item 90, comprising two or more types of antibodies selected from: or an antigen-binding portion thereof.
Item 92. In another embodiment, when TF-1 cells proliferate upon induction of hGM-CSF, two or more antibodies, or antigen-binding portions thereof, inhibit TF-1 cell proliferation by 80% or more at a concentration of about 55 pM each. This is a method for enhancing the activity disclosed in item 91.
Item 93. Another embodiment is a pharmaceutical or veterinary pharmaceutical composition comprising multiple types of anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof that are specific for the same specific antigen and a pharmaceutically acceptable carrier. is there.
Item 94. In another embodiment, the plurality of types of anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof are the following (a) and (b):
(A) It has a complementarity determining region (CDR) represented by the amino acid sequence of SEQ ID NO: 4 to 9, SEQ ID NO: 330 to 335, SEQ ID NO: 336 to 341, or SEQ ID NO: 342 to 347, and is specific for hGM-CSF An anti-hGM-CSF monoclonal antibody or an antigen-binding portion thereof,
(B) the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof of (a) having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added, and specific for hGM-CSF
The pharmaceutical composition or veterinary pharmaceutical composition disclosed in item 93, comprising two or more kinds of antibodies selected from the group consisting of antibodies or antigen-binding portions thereof.
Item 95. In another embodiment, when TF-1 cells proliferate upon induction of hGM-CSF, said two or more antibodies or antigen binding portions thereof inhibit TF-1 cell proliferation by 80% or more at a concentration of about 55 pM each. The pharmaceutical composition or veterinary pharmaceutical composition disclosed in item 94, wherein

  Examples of the present invention will be described more specifically below, but the examples are not intended to limit the scope of the present invention.

Isolation of cell clones that produce fully human antibodies to hGM-CSF (hGM-CSF) FIG. 1 shows a flowchart of isolation of antibody producing cell clones. B lymphocytes were isolated from blood of donors with high serum anti-GM-CSF monoclonal antibody titers and then infected with EBV. Cells grown by infection with EBV were used as a library for antibody-producing cells.

  The antibody-producing cell library cells were dispersed on a 96-well plate and cultured for 3 to 4 weeks. The culture supernatant of each well was screened for the presence of anti-hGM-CSF monoclonal antibody. Screening was performed by ELISA using 96 well plates coated with recombinant hGM-CSF (rhGM-CSF). Cells in wells positive for antibody production are seeded onto a new 96-well plate and cultured for 3 to 4 weeks, and then the culture supernatant of each well is added to a second anti-hGM-CSF monoclonal antibody presence. Screened. Cells in wells positive for antibody production were subjected to ultradilution culture on 96-well plates for 3 to 5 weeks. Thereafter, the culture supernatant of each well was tested for the presence of antibody, and clones of antibody producing cells were finally obtained from wells plated at 1 cell per well.

Identification of antibody isoforms and subclasses The antibody isoforms and subclasses produced by the three antibody producing cell clones EV1007, EV1018, and EV1019 were identified (Table 1). Identification is performed by ELISA, which uses the culture supernatant as the first antibody and isotype-specific and subtype-specific antibodies as the second antibody to screen for antibody production in the culture supernatant. It was essentially the same as used.
Table 1 shows the names of the antibodies and their subclasses against the three newly obtained anti-hGM-CSF monoclonal antibodies and the monoclonal antibody J1584C (hereinafter referred to as EV1003) reported in International Publication No. WO07 / 049472. It is.

table 1

Cloning of antibody genes from antibody producing cells Antibody genes were cloned from antibody producing cells. Total RNA was extracted from antibody producing cells and its cDNA was synthesized using oligo dT primer and reverse transcriptase.
By using cDNA as a template, the gene encoding the human antibody was amplified by PCR. Primers were designed based on a database of DNA sequences of antibody genes so that the 5 ′ end contains a transcription initiation site and the 3 ′ end contains a translation termination site.

Determination of the amino acid sequence of an antibody based on the nucleotide sequence cDNAs of antibody genes [EV1007, EV1018, and EV1019, each consisting of heavy (H) chain and light (L) chain genes] were cloned into a plasmid vector and these nucleotides The sequence was analyzed by a sequencer (ABI). These amino acid sequences were determined based on the nucleotide sequences obtained. The Kabat method was used for analysis of the complementarity determining regions (CDRs) of the antibodies (EV1007, EV1018, EV1019, and EV1003). The CDR sequences of the four antibodies are represented by SEQ ID NOs: 4-9 and 330-347. Specifically, L chain CDR1, L chain CDR2, L chain CDR3, H chain CDR1, H chain CDR2, and H chain CDR3 of EV1018 are shown in SEQ ID NOs: 4 to 9, respectively. EV1019 L chain CDR1, L chain CDR2, L chain CDR3, H chain CDR1, H chain CDR2, and H chain CDR3 are shown in SEQ ID NOs: 330 to 335, respectively. The L chain CDR1, L chain CDR2, L chain CDR3, H chain CDR1, H chain CDR2, and H chain CDR3 of EV1003 are shown in SEQ ID NOs: 336 to 341, respectively. The L chain CDR1, L chain CDR2, L chain CDR3, H chain CDR1, H chain CDR2, and H chain CDR3 of EV1007 are shown in SEQ ID NOs: 342 to 347, respectively.

Confirmation of the obtained antibody gene encoding anti-hGM-CSF monoclonal antibody Genes encoding three antibodies (each consisting of H and L chains) were inserted into an expression vector. Plasmids encoding the L and H chain genes of each antibody were transiently transfected into 293T cells using Lipofectamine and Plus reagent (Invitrogen) and tested for transient antibody expression.
Two days after transfection, the cell culture supernatant containing the secreted antibody was collected. Human IgG and anti-GM-CSF monoclonal antibody in the culture supernatant were detected by ELISA to confirm the transient expression of human antibody and anti-GM-CSF monoclonal antibody.

Establishment of stable CHO-transfectant cells expressing anti-hGM-CSF monoclonal antibodies Expression vectors encoding anti-GM-CSF monoclonal antibodies were transfected into CHO-K1 cells as described above. Two days after transfection, the cells were seeded in 96-well plates and cultured for about 2 weeks in selective medium containing the appropriate selectable marker. The culture supernatant of each well was screened for anti-hGM-CSF monoclonal antibody by ELISA. One cell in a well positive for antibody expression was plated into a well of a 96 well plate. One cell clone grown in the selective medium was screened for the expression of anti-GM-CSF monoclonal antibody to obtain a cell clone stably expressing the anti-GM-CSF monoclonal antibody.

Antibody Purification A CHO cell clone stably expressing the anti-GM-CSF monoclonal antibody was cultured in serum-free medium. After each expression period, the culture supernatant was collected and the antibody was isolated by affinity chromatography using a HiTraprProteinA FF prepack column (Amersham) according to the manufacturer's instructions. It was confirmed that the purified antibody had binding activity to hGM-CSF by ELISA and consisted of an antibody of about 50 kDa H chain and about 25 kDa L chain by SDS-PAGE.

Affinity analysis of anti-GM-CSF monoclonal antibody To calculate the affinity constant for binding of recombinant hGM-CSF to anti-hGM-CSF monoclonal antibody, surface plasmon resonance (SPR) was performed on Biacore System ™. Performed (FIG. 2).
In the method for analyzing the interaction between antibody and antigen, purified antibody is captured on the sensor chip by interaction with protein G immobilized on the surface of the sensor chip, and then the recombinant antigen is applied to the sensor chip. Injected. Using purified antibody, recombinant hGM-CSF was used as the antigen.
The equilibrium dissociation constants (K _D ) for yeast-derived (Leukine Berlex) and E. coli-derived (Peprotech) recombinant hGM-CSF against anti-hGM-CSF monoclonal antibodies are shown in Table 2.

Table 2

In this assay, K _D values for yeast GM-CSF (Leukine) is a 1.2 × 10 In EV1007 ^-9 M, the EV1018 2.3 × 10 ^-10 M, the EV1019 3.6 × 10 ^-10 M there were. The K _D value for the previously produced monoclonal antibody EV1003 was 2.3 × 10 ⁻¹⁰ M.
E. coli K _D values for (Peprotech) is, EV1007 in 9.3 × 10 ^-10 M, the EV1018 1.5 × 10 ^-10 M, the EV1019 1.5 × 10 ^-10 M, EV1003 in 9.5 × 10 ^-11 M. All four hGM-CSF monoclonal antibodies produced bound to recombinant hGM-CSF with high affinity.

Inhibitory Effect of Anti-GM-CSF Monoclonal Antibody on Peripheral Blood Dendritic Cell Proliferation 5 × 10 ⁵ dendritic cells (DC) including plasmacytoid DC and myeloid DC were transformed into Blood Dendritic Cell Isolation Kit II Human (Miltenyi Biotech). ) From 7 × 10 ⁷ human mononuclear cells.
The resulting DCs were in RPMI 1640 medium (Gibco) supplemented with 10% FCS, rhGM-CSF (1 ng / mL, Peprotech), TNF-α (10 ng / mL), and anti-GM-CSF monoclonal antibody (1 μg / mL). And inoculated into a 96-well flat bottom plate at a concentration of 2 × 10 ⁴ cells / well and incubated for 10 days.
Anti-GM-CSF polyclonal antibody (anti-GM-CSFpAb, R & D) (1 μg / mL) and human anti-human cytomegalovirus monoclonal antibody (hIgG) (1 μg / mL) produced by the inventors were used as controls. The results are shown in FIG.
As seen in FIG. 3, EV1003, EV1018, and EV1019 inhibited GM-CSF dependent proliferation of DC. In contrast, EV1007 showed no inhibition. On the other hand, anti-GM-CSFpAb inhibited GM-CSF-dependent proliferation of DC, but hIgG did not.
EV1003, EV1018, and EV1019 were able to independently block GM-CSF dependent proliferation of DC.

Evaluation of Neutralizing Potency of Anti-GM-CSF Monoclonal Antibody Using TF-1 Cells As shown in FIGS. 4 and 5, the present inventors used TF-1 cells that proliferate depending on the presence of hGM-CSF. The neutralizing ability of purified anti-hGM-CSF monoclonal antibodies (EV1007, EV1018, and EV1019) and EV1003, previously produced using the same, were tested.
Purified antibody was preincubated with recombinant hGM-CSF and the mixture was added to TF-1 cell culture. After 2 days in culture, the proliferation state of TF-1 cells was measured by colorimetric analysis. Antibodies with GM-CSF neutralizing activity were able to block added recombinant GM-CSF activity, resulting in inhibition of GM-CSF dependent TF-1 cell growth.
4 and 5 show the inhibitory effect (neutralizing activity) of the antibody on the growth of TF-1 stimulated by yeast hGM-CSF (Leukine) and Escherichia coli hGM-CSF (Peprotech), respectively.

Each antibody was independently assessed for its inhibitory effect, and anti-GM-CSFpAb and hIgG were used as controls as described in Example 9.
Antibodies diluted 4-fold in a concentration range from 2 μg / mL to 31 pg / mL were tested. A final concentration of 0.5 ng / mL rhGM-CSF was used for all tests. After 40 hours of incubation, the viability of TF-1 cells was measured using A450 / ref. Evaluation was based on the color intensity of A495.
In the y-axis of FIGS. 4 and 5, the survival rate of TF-1 cells with 31 pg / mL control hIgG was set to 100%, and the survival rate of TF-1 cells without rhGM-CSF was set to 0%. . The antibodies tested are shown at the bottom of the figure and their concentrations are shown on the right side of the figure.

The growth of TF-1 cells was stimulated using yeast rhGM-CSF (Leukine) at a concentration of 0.5 ng / mL, and the results of the neutralizing ability of each of the anti-hGM-CSF monoclonal antibodies are shown in FIG. HIgG as a negative control exhibited non-specific inhibition of cell growth at concentrations as high as 2 μg / mL, but showed no inhibition at concentrations below 2 μg / mL. Anti-hGM-CSF polyclonal antibody (anti-GM-CSFpAb) showed cell growth inhibition depending on antibody concentration above 125 ng / mL or 125 ng / mL.
Among the purified monoclonal antibodies, EV1003 inhibits the growth of TF-1 cells to a concentration of 31 ng / mL up to 60% of the positive control, with concentration-dependent growth inhibition exceeding 31 ng / mL or 31 ng / mL. Observed. EV1018 and EV1019 showed about 50% inhibition at concentrations above 0.5 ng / mL or 0.5 ng / mL and at concentrations above 2 ng / mL or 2 ng / mL, respectively. It was confirmed that EV1018 and EV1019 had particularly high neutralizing activity against hGM-CSF. EV1007 shows clear cell growth inhibition at concentrations of 0.5 μg / mL or above 0.5 μg / mL, indicating that its neutralizing capacity is very low.

The growth of TF-1 cells was stimulated using E. coli rhGM-CSF (Peprotech) at a concentration of 0.5 ng / mL, and the results of the neutralizing ability of each of the anti-hGM-CSF monoclonal antibodies are shown in FIG. In this assay, hIgG as a negative control exhibited non-specific inhibition of cell growth at a high concentration of 2 μg / mL, but showed no inhibition at concentrations below 2 μg / mL. Anti-hGM-CSF polyclonal antibody (anti-GM-CSFpAb) showed antibody concentration-dependent cell growth inhibition above 31 ng / mL or 31 ng / mL.
Among the purified monoclonal antibodies, EV1003 showed a clear inhibition of TF1 cell proliferation in a concentration dependent manner over 31 ng / mL or over 31 ng / mL. EV1018 and EV1019 showed greater than 50% inhibition at concentrations above 0.5 ng / mL or 0.5 ng / mL, and at concentrations above 2 ng / mL or 2 ng / mL, respectively. It was confirmed that the neutralizing activity of EV1018 and EV1019 was particularly high.

EV1007 clearly showed cell growth inhibition at concentrations of 0.5 μg / mL or above 0.5 μg / mL, indicating that its neutralizing ability was very low compared to EV1018 and EV1019.
The neutralizing activity of the combination of the two antibodies was evaluated (FIGS. 6 and 7). The five combinations of antibody mixtures are listed in Table 3.

４μｇ／ｍＬから６２ｐｇ／ｍＬまでの濃度範囲の４倍段階希釈によって各抗体用の試験溶液を調製し、同じ濃度の抗体２つを混合した（最終濃度を図６および図７に示す）。ＴＦ−１細胞の生存率を、Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ（ＷＳＴ−１アッセイ、ＤＯＪＩＮ）を用いて、Ａ４５０／ｒｅｆ．Ａ４９５の発色強度によって評価した。 Table 3

Test solutions for each antibody were prepared by 4-fold serial dilution in the concentration range from 4 μg / mL to 62 pg / mL, and two antibodies of the same concentration were mixed (final concentrations are shown in FIGS. 6 and 7). The viability of TF-1 cells was measured using the Cell Counting Kit (WST-1 assay, DOJIN) using A450 / ref. Evaluation was based on the color intensity of A495.

  In the y-axis of FIGS. 6 and 7, the survival rate of TF-1 cells with 31 pg / mL control hIgG was set to 100%, and the survival of TF1 cells without rhGM-CSF as shown in FIGS. The rate was set to 0%. The results of TF1 cell viability with 31 pg / mL control hIgG are shown in FIGS. 4 and 5, but not in FIGS. The mixed antibody combinations are shown at the bottom of the figure, and their concentrations are shown on the right side of the figure.

Among these combinations, the inhibitory effects of Mix-2: EV1003 + 1007, Mix-3: EV1003 + 1018, and Mix-4: EV1003 + 1019 were particularly increased compared to the single use of each antibody. For example, as seen in FIG. 6, Mix-4: EV1003 + 1019 inhibited TF1 cell growth to 10% of the positive control at each antibody concentration of 8 ng / mL (approximately 55 pM).
As shown in FIG. 4, EV1003 alone showed no inhibition of TF1 cell proliferation at a concentration of 8 ng / mL, and EV1019 alone showed only 50% inhibition at a concentration of 8 ng / mL. These results indicated that the combination of the two antibodies exhibited a particularly high neutralizing activity.
Although the inhibitory effect of EV1007 when used alone is very low, Mix-2: EV1003 + 1007 inhibits TF1 cell growth to 10% of the 8 ng / mL positive control at each antibody concentration, as shown in FIGS. (About 55 pM).

As shown in FIG. 7, when E. coli rhGM-CSF (Peprotech) was used to stimulate the growth of TF-1 cells, the growth of the cells was as follows: Mix-2: EV1003 + 1007 with each concentration exceeding 31 ng / mL or 31 ng / mL , Mix-3: EV1003 + 1018, and Mix-4: EV1003 + 1019. It has been confirmed that when antibodies are added in combination, they exhibit particularly strong neutralizing activity.
On the other hand, the inhibitory effect of Mix-5: EV1018 + 1019 was increased in a concentration-dependent manner as compared to the antibody used alone as shown in FIGS. 4 and 5, but the increase in the effect was higher than that of Mix-2 to 4 Was also low.
Mix-5: EV1018 + 1019 had no significant effect of mixing, and the dose-dependent curves (neutralization effect pattern) of both antibodies used alone were similar.
As shown in FIGS. 4 and 5, among the four antibodies (EV1003, EV1007, EV1018, and EV1019), EV1003 showed the strongest inhibition when used alone at a concentration of 2 μg / ml. But about 20% of the cells remained. In contrast, Mix-2, Mix-3, and Mix-4 almost completely inhibited cell growth in the concentration range from 31 ng / mL to 125 ng / mL. It was pointed out that these three combinations (Mix-2, Mix-3, and Mix-4) exhibited particularly high neutralizing activity.

Mix-1, which is a combined use of anti-GM-CSF monoclonal antibody (EV1003) and anti-CMV antibody (hIgG), did not enhance the inhibitory effect of the antibody compared to the use of these alone.
Of the three antibodies obtained in the present invention, two antibodies (EV1018 and EV1019) showed particularly high neutralizing activity when used alone and in combination. Of the antibody combinations listed in Table 3, some combinations exhibited higher neutralizing activity than the additive action of both antibodies.
The anti-GM-CSF monoclonal antibodies produced as described above or antigen-binding portions thereof can specifically bind to hGM-CSF causing various diseases, and inactivate (neutralize the physiological activity of hGM-CSF. )can do. Thus, the antibody or antigen-binding portion thereof has a higher neutralizing activity against hGM-CSF than the anti-hGM-CSF monoclonal antibody prepared so far. Since the antibody or antigen-binding portion thereof is a human monoclonal antibody, it does not exhibit immunogenicity and does not elicit an immune response.

Furthermore, the simultaneous combined use of anti-GM-CSF monoclonal antibodies or their antigen binding portions exhibits a particularly high inhibitory effect (ie neutralizing activity) on cell growth compared to their single use.
In view of these properties, the anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof described in the present invention are asthma, COPD, cystic fibrosis, intestinal lung disease, rhinitis, atopy, and GM- including allergic diseases such as hay fever, graft rejection, graft-versus-host disease (GVHD), arthritis and related joint diseases, psoriasis, myeloid leukemia, multiple sclerosis, Alzheimer's disease, and rheumatoid arthritis Although effective as a prophylactic or therapeutic agent at low doses for diseases associated with elevated CSF, the effect is not limited to these diseases.

Affinity measurement of anti-GM-CSF monoclonal antibody in Biacore hGM-CSF Each antibody was immobilized using a mouse anti-human Biacore chip (GE Healthcare), and soluble commercially available GM-CSF (Biomol) was bound thereto. The hGM-CSF used in these experiments is the same as that used in the bioassay described below. The binding affinity was determined using Biacore software and the results are summarized in Table 4.

ＥＶ１０１８の結合親和性はＥＶ１０１９より高く、ＥＶ１００３よりも非常に高い。ＥＶ１０１８の変種は全て、野生型ＥＶ１０１８に等しい結合親和性を有する。 Table 4

The binding affinity of EV1018 is higher than EV1019 and much higher than EV1003. All variants of EV1018 have a binding affinity equal to wild type EV1018.

Cross-reactivity with GM-CSF from other species (Rhesus monkey, mouse, marmoset) Rhesus monkey GM-CSF sequences are present in public databases (eg GenBank accession number NP_001028121) Used for designing. A protease cleavage site is introduced to release untagged rhesus GM-CSF protein. The resulting protein has excellent biological activity (see next section) and is recognized by all anti-GM-CSF monoclonal antibodies (EV1018, EV1019, EV1003) in Western blots (data is Not shown). Biacore measurements (using the same setup as described in 1.1 above) were made and the results are shown in Table 5.

Table 5. Rhesus monkey GM-CSF monoclonal anti-GM-CSF monoclonal antibody binding test

  In all cases, cross-reactivity with rhesus monkey GM-CSF is excellent (all anti-GM-CSF monoclonal antibodies recognize rhesus monkey GM-CSF protein with a factor roughly 2 to 3 lower than hGM-CSF). . In addition, rhesus monkeys provide an excellent non-human primate animal model for the development of the disclosed anti-GM-CSF monoclonal antibodies.

  Measurements were performed with mouse GM-CSF (Pepro Tech and Biomol; eg GenBank accession number NP — 034099) and the results are summarized in Table 6.

全ての場合において、マウスＧＭ−ＣＳＦとの交差反応性は非常に弱い。このデータは、抗ＧＭ−ＣＳＦモノクローナル抗体が結合するｈＧＭ−ＣＳＦエピトープを同定することを目的とした実験に対する基礎を提供するものであり、マウスとヒトの間のＧＭ−ＣＳＦタンパク質のハイブリッドが確立されている。 Table 6. Mouse GM-CSF monoclonal anti-GM-CSF monoclonal antibody binding test

In all cases, the cross-reactivity with mouse GM-CSF is very weak. This data provides the basis for experiments aimed at identifying hGM-CSF epitopes bound by anti-GM-CSF monoclonal antibodies, and a GM-CSF protein hybrid between mouse and human was established. ing.

  A marmoset GM-CSF expression clone similar to the rhesus monkey GM-CSF expression clone described above (eg, GenBank accession number B0KWQ4 with methionine at position 53 and proline at position 109) is prepared. The purified protein was used for Biacore measurements and the results are summarized in Table 7.

マーモセットＧＭ−ＣＳＦとの交差反応性は著しい。抗ＧＭ−ＣＳＦモノクローナル抗体は全て、ｈＧＭ−ＣＳＦよりも大まかに１８倍から１９倍劣るファクターでマーモセットＧＭ−ＣＳＦタンパク質を認識する。アカゲザルの場合におけるように、マーモセットは、前記開示した抗ＧＭ−ＣＳＦモノクローナル抗体の開発のための、優れた動物モデルを提供する。 Table 7. Marmoset GM-CSF anti-GM-CSF monoclonal antibody binding test

The cross-reactivity with marmoset GM-CSF is remarkable. All anti-GM-CSF monoclonal antibodies recognize marmoset GM-CSF protein with a factor that is roughly 18 to 19 times inferior to hGM-CSF. As in the case of rhesus monkeys, marmoset provides an excellent animal model for the development of the disclosed anti-GM-CSF monoclonal antibodies.

Biological activity: neutralization of GM-CSF bioactivity TF-1 proliferation assay (GM-CSF of human, rhesus monkey, marmoset)
The proliferation of the TF-1 human cell line is growth factor dependent. This cell line is known in the art as an excellent basis for testing for the biological effects of growth factors.

  Here, (i) recombinant hGM-CSF (rhGM-CSF) of hGM-CSF expressed in E. coli (Fa. Biomol # 2514) by using standard techniques well known in the art, or (Ii) recombinant rhesus monkey GM-CSF (rrGM-CSF) or (ii) marmoset GM-CSF (eg, methionine at position 53, proline at position 109), rhesus monkey GM-CSF (eg, GenBank Accession No. Various species such as recombinant marmoset GM-CSF (rmGM-CSF) of GenBank accession number B0KWQ4) (both expressed in HEK293 using standard techniques well known in the art) Recombinant G against -CSF used (rGM-CSF).

  TF-1 cells were cultured in cell culture medium (RPMI1640 (Cat. 31870 Fa. Gibco), 1 × Glutamax 100 × (Cat. 35050-038 Fa. Gibco), 1 mM Pyrobat sodium (Sodium Pyrovat) 100 mM (Cat. )) Grow in 10 mM Hepes 1M (Cat. 15690-056 Fa. Gibco), 10% FCS (Cat. 10500-064 Fa. Gibco), 2 ng / ml rGM-CSF (Cat. 200-005LFa. ReliaTech GmbH). Cells are washed 3 times with PBS and seeded in 96-well plates (Cat. 167008 Fa. Nunc) in assay medium (cell culture medium without rGM-CSF) at a concentration of 1E5 cells / ml. The antibody solution and GM-CSF solution are diluted in dilution medium (cell culture medium without FCS and without rGM-CSF) and 10 μl volumes are added each. Cells are incubated for 3 days at 37 ° C. and 5% CO 2 in a humidified chamber. The cell viability is measured by adding 20 μl MTS and measuring the absorbance at 492 nm according to the instructions of the kit (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Fa. Promega, Cat. No. G3581). The negative control is TF-1 cells grown in cell culture medium without rGM-CSF. The positive control is TF-1 cells grown in cell culture medium containing an ED80 concentration of rGM-CSF.

Prior to testing the neutralizing activity of the antibodies, ED80 for stimulating proliferation of human, rhesus monkey, and marmoset on GM-CSF was determined (Table 8). Although human and rhesus GM-CSF can stimulate TF-1 proliferation very effectively (ED _{80 [human]} : 1.5 ng / ml, ED _{80 [rhesus monkey]} : 7 ng / ml), Marmoset GM-CSF is a somewhat ineffective stimulator of TF-1 proliferation. Thus, human cell line TF-1 is suitable for testing neutralizing activity against human and rhesus monkey GM-CSF, but not for testing marmoset-derived GM-CSF.

抗体のＩＣ５０測定を、ＥＤ８０濃度下で行う。
図８Ａおよび図８Ｂのｙ軸において、陽性対照のＴＦ−１細胞の生存率を１００％に設定し、陰性対照のＴＦ−１細胞の生存率を０％に設定した。 Table 8. ED80 for human, rhesus monkey, and marmoset GM-CSF in TF-1 proliferation assay

Antibody IC50 measurements are performed under ED80 concentrations.
On the y-axis of FIGS. 8A and 8B, the viability of the positive control TF-1 cells was set to 100% and the viability of the negative control TF-1 cells was set to 0%.

A commercially available rat anti-hGM-CSF IgG2a antibody with known neutralizing activity is also used (BVD2-23B6, Fa.BD Pharmingen # 554501) (Table 9). The isotype control antibody rat IgG2a (R35-95, Fa. BD # 554687) showed no inhibition of cell proliferation (data not shown).
EV1018 and EV1019 had excellent neutralizing activity of human recombinant GM-CSF. EV1018 exceeds the neutralizing activity of BVD2-23B6.
The heavy chain of EV1018 naturally contains an N-glycosylation site within framework 3. This is shown, for example, in SEQ ID NO: 10, corresponding to residues 95-97. Therefore, a variant of EV1018 heavy chain was produced and tested for its neutralizing activity. As shown in Table 9, the results indicate that EV1018 bioactivity is maintained after removal of the N-linked glycosylation site in heavy chain framework 3. Table 9 shows the IC50 values for two glycosylation site sequence variants (EV1018 variants 1 and 2) designated N95K and T97A, respectively, compared to the WT EV1018 heavy chain containing the N-glycosylation site. IC50 values for each purified IgG were measured in the TF-1 assay essentially as described. Experiments with additional EV1018 variants yielded similar results that were comparable to those of EV1018.

Table 9. Comparison of IC50 in TF-1 proliferation assay expressed in ng / ml and pM (assuming a molecular weight of 150 kDa for the antibody)

  In experiments using recombinant rhesus monkey GM-CSF monoclonal antibodies, EV1018, EV1019, and EV1003 have much higher neutralizing activity than BVD2-23B6 from rats. However, in both types of experiments, EV1018 is significantly superior across all tested antibodies. All variants of EV1018 equally neutralize the activity of wild type EV1018.

  In the experimental set-up that resulted in the results shown in Table 9, commercial rat anti-human GM- can not be obtained under a given ED80 concentration, i.e., a dose of rGM-CSF of 1.5 ng / ml. The use of CSF IgG2aBVD2-21C11 (Fa.BD, # 554503) is not possible. To measure antibody 21C11, lower doses of rGM-CSF are required, as shown in Table 10 below.

  Comparison of antibody potency in different laboratories is not possible by comparing IC50 values alone, since IC50 values are highly dependent on assay conditions such as the amount of stimulation used. By comparing ratios such as IC50 values obtained with the TF-1 assay described above, a much more reliable comparison between the standard and subject antibodies at various rGM-CSF doses as indicated Is possible. For example, WO 2006/122797 (pages 56 and 57, Table 8) disclosed that its best antibody is 35 times better than the commercially available antibody, rat anti-human GM-CSF, clone BVD2-21C11. The ratio between the IC50s between the commercially available anti-human GM-CSF monoclonal antibody clones BVD2-21C11 and EV1018 is 413. Therefore, based on comparison to commercial standards, the neutralizing activity of EV1018 is about 10 times higher as shown in Table 10. This is surprising since WO 2006/122797 teaches those skilled in the art that the neutralizing activity of anti-GM-CSF monoclonal antibodies strongly correlates with the binding affinity for GM-CSF. (WO 2006/122797, improvement in binding affinity shown in Table 4 on page 50 correlates with improvement in neutralization potency shown in Tables 6 and 7 on pages 52-54).

  Compared to the binding affinity of EV1018 (WO 2006/122797, page 50, Table 4: binding affinity is 7 pM for MOR04357-Fab) we compared the binding affinity of EV1018 to GM-CSF. It was understood that the binding was about 16 times lower than MOR04357. However, the neutralizing activity of EV1018 is 10 times higher than that of MOR04357, indicating that the neutralizing activity does not necessarily correlate with the binding affinity for GM-CSF.

Table 10. Relative neutralizing activity of EV1018 TF-1 assay and prior art antibodies
1st and 2nd lines: WO2006 / 122797, pages 56 and 57, data from Table 8, 3rd and 4th lines: our data

IL-8 secretion assay in U937 cells (human versus rhesus GM-CSF)
IL-8 is a pro-inflammatory cytokine. This is a critical factor for neutrophil inflammation and is involved in most inflammatory responses. Target cells for GM-CSF activity are cells of the myeloid lineage. The promonocytic cell line U937 is derived from bone marrow cells and secretes the pro-inflammatory cytokine IL-8 upon stimulation with GM-CSF.

U937 (ATCC: CRL-1593.2) was added to cell culture medium (RPMI1640 (Cat. 31870 Fa. Gibco), 1 × Glutamax, 100 × (Cat. 35050-038 Fa. Gibco), 10% FCS (Cat. 10500-064 Fa.). Gibco) and grown in 1% PenStrep). Inoculate 100 μl of 1E5 cell / ml cell suspension and add 10 μl of antibody solution and 10 μl of GM-CSF solution. Cells are incubated for 24 hours at 37 ° C. and 5% CO ₂ in a humidified chamber. IL-8 cytokine levels are measured using an OptEIA human IL-8 Elisa Set (Fa. BD Bioscience, Cat. No. 555244).

  Negative control is U937 cells grown in cell culture medium without addition of antibody and without addition of GM-CSF, and positive control is in cell culture medium containing ED80 rGM-CSF without addition of antibody solution. U937 cells grown in The ED80 concentration for human and rhesus monkey GM-CSF is measured (Table 9) and this concentration is used to determine the IC50 for various anti-GM-CSF monoclonal antibodies (Table 11).

Table 11. Measurement of ED80 in IL-8 secretion in U937

Table 12. Comparison of IC50 in U937 IL-8 secretion assay. Results are expressed in ng / ml and pM (assuming a molecular weight of 150 kDa for the antibody).

  In this cellular assay, all three antibodies neutralized human GM-CSF and rhesus GM-CSF very efficiently. EV1018 has a higher neutralizing activity than EV1019 and much higher neutralizing ability than EV1003. All variants of EV1018 exhibit neutralizing activity equal to wild type EV1018.

Induction of surface CD11b / Mac1 Granulocytes are a subpopulation of myeloid cells and are target cells for GM-CSF bioactivity. In addition to other effector functions, GM-CSF induces the adhesion molecule CD11b / Mac1 on the surface of granulocytes. Induction of CD11b / Mac1 on the surface of cells of the myeloid lineage is an essential step in cell migration from peripheral blood into inflamed tissues. In patients with chronic inflammatory airway diseases such as COPD and asthma, the number of granulocytes in sputum and bronchoalveolar lavage fluid is increased. Therefore, the efficacy of anti-GM-CSF antibodies was tested against GM-CSF mediated induction of adhesion marker CD11b / Mac1 on primary h granulocytes.

  Anti-GM-CSF monoclonal antibody or isotype antibody (human IgG, # I- 2511; Fa. Sigma-Aldrich) (positive control) for 15 minutes at 37 ° C. The negative control is a clot-inhibited blood incubated with isotype antibody preincubated with PBS, 01% BSA for 20 minutes at 37 ° C. for 15 minutes.

To quantify CD11b / Mac1 expression, an anti-CD11b antibody labeled with phycoerythrin (Fa: Pharmingen; Cat: 333142) is used according to the supplier's instructions. Briefly, 20 μl of anti-CD11b antibody solution is added to the cells and incubated for 30 minutes at room temperature in the dark. Red blood cells (erythrocytes) are lysed by adding 2 ml of Lysis solution (Fa: BD; Cat349202) and incubated at room temperature in the dark for an additional 10 minutes. After two washing steps with PBS, 0.1% BSA, leukocytes are suspended in 500 μl of Cellfix (Fa: BD; Cat340181). The analysis uses an LSRII flow cytometer and DIVA 6.1.1 software for FACS data analysis (both Fa.BD). Granulocyte cell populations are gated on a dot plot using forward and lateral scatter and set to 100%. rhGM-CSF 30 pM is used to stimulate CD11b / Mac1 on the surface of granulocytes. EV1018 67pM is sufficient to completely block GM-CSF-induced CD11b / Mac1.
These data indicate that EV1018 effectively neutralizes the bioactivity of GM-CSF in situations that are highly significant for inflammatory processes with increased granulocyte counts such as COPD and asthma. ing.

Pulmonary inflammation after tobacco smoke exposure in C57BL / 6J mice Tobacco smoke is the most important factor in developing COPD. Long-term exposure to animal species such as rats and mice causes many pathophysiologically relevant anatomical lesions comparable to human disease (Fujita and Nakanishi, 2007). One important parameter of COPD is chronic bronchitis, which includes an increase in the number of neutrophils and macrophages in the lung. To demonstrate that GM-CSF plays a critical role in smoke-induced lung cell influx, an anti-GM-CSF monoclonal antibody was used in a long-term tobacco smoke model in mice.
Mice (C57BL / 6J strain, 18-23 g) were exposed to tobacco smoke for 4 days. Mice were exposed to 6 cigarettes on days 1 and 2, 8 cigarettes on day 3, and 10 cigarettes on day 4. Each tobacco exposure lasted 16 minutes and was then exposed to fresh air for 8 minutes. An additional break 24 minutes was performed with exposure to tobacco every second and exposure to fresh air. Mice not exposed to cigarette smoke served as negative controls.

Rat anti-mouse GM-CSF IgG2a, 300 μg (clone MPI-22E9, Fa.eBioSciences, # 16-7331) or a suitable isotype antibody, 300 μg (rat IgG2a, Fa.eBioSciences, # 16-4321) or vehicle 300 μl (PBS 10 mM NaCl) was administered intraperitoneally (ip) 2 days prior to the smoke protocol on days 1 and 3. 18 hours after the last exposure, the animals were euthanized and the lungs were lavaged with 2 × 0.8 ml Hanks solution containing EDTA. Bronchoalveolar lavage fluid (BALF) was separated into cell pellets and supernatant by centrifugation. Myeloperoxidase (MPO) activity in the pellet was measured as a parameter for bone marrow cell influx. A 500 μl wash sample was spun down at 485 × g for 10 minutes at 4 ° C. The pellet was resuspended in 200 μl of 0.5% hexadecyltrimethylammonium bromide (HTAB). 50 μl of the suspension was transferred to a 96 well microtiter plate. 250 μl of substrate solution (50 mM KH ₂ PO ₄ , 5 mM Na ₂ PO ₄ , 0.2 mg / ml o-dianisidine dihydrochloride, 0.2 μl / ml H ₂ O ₂ ) was added to each well to initiate the enzyme reaction. . The disappearance at 450 nm was measured for 90 seconds. Myeloperoxidase (MPO) activity was calculated during the steady state of the enzyme reaction. Activity is expressed as milliactivity units per minute (mAU / min).

Cytokines MCP-1 and MIP-1α were measured by detecting multiple analytes in a BALF supernatant on a flow cytometer (LSRII, Becton Dickinson). The assay was performed according to instructions from Bender MedSystems (Flow Cytomix). Briefly, 25 μl of BALF supernatant was replaced with 25 μl of 1 × assay buffer (Basic Kit BMS8440FF), Bead Mixture (mixture of MCP-1 and MIP-1a, Simplex Kits, BMS8605FF, BMS86013FF), biotin conjugate mixture (Simplex Kit) And incubated for 2 hours at room temperature on a 500 rpm microplate shaker. After washing twice with assay buffer, the beads were dissolved in 100 μl of assay buffer and 50 μl of streptavidin-PE-solution (included in Basic Kit) was added. The beads were incubated for 1 hour at room temperature on a 500 rpm microplate shaker. The beads were washed twice with assay buffer. The beads were dissolved in 500 μl of assay buffer and analyzed by flow cytometry using FlowCytomix Pro2.2 software supplied by Bender MedSystems. For quantification, standard dilutions (included in the Simple Kit) were prepared according to the instructions of the supplier (Bender MedSystems). Cytokine concentrations were determined based on standards.
When mice were pretreated with anti-GM-CSF antibody, MPO activity was reduced by approximately 25% (FIG. 9A). Consistent with this result, the cytokines MCP-1 and MIP-1a were reduced by approximately 18% and 23%, respectively (FIG. 9B).
The results shown in FIGS. 9A and 9B show that GM-CSF plays a central role in tobacco smoke-induced inflammation, and neutralization of GM-CSF is induced by cellular infiltration and prolonged tobacco smoke exposure in mice. That inflammatory cytokines can be significantly reduced.

hGM-CSF epitope mapping To observe whether a particular linear sequence (s) in GM-CSF can be identified as an epitope recognized by the antibody in question, mouse, human, rhesus monkey, And peptide arrays containing marmoset GM-CSF peptides were tested.
The following antibodies were tested: BIBH1 (an irrelevant humanized IgG1, FAP-recognizing negative control antibody (US 20030103968, Use of FAP alpha specific antibody BIBH1 in the treatment of cancer (http://cow.as/Sw/ / Abstracts + & + Virtual + Meeting / Abstracts? & Vmview = abst_detail_view & confID = 10 & abstractID = 1028)), EV1018, EV1019, and EV1003.

  Results from the peptide array indicate that the negative control antibody did not show any specific binding above the level, but the immobilized GM-CSF and human IgG positive controls gave robust signals. It was. Tested anti-GM-CSF antibodies are non-linear or conformation-specific (one or more) since properly folded GM-CSF is recognized, but not GM-CSF derived peptides. We conclude that we have recognized the epitope. This is in contrast to some other anti-GM-CSF antibodies known in the literature (eg WO2007 / 092939).

The following method was used: custom arrays were synthesized and printed on glass slides at JPT (Berlin, Germany). The array was blocked with PBS + 1% BSA + 0.1% Tween 20 for 3 hours at room temperature with gentle rocking. The antibody is diluted to 5 μg / ml in probing buffer (PBS, 5 mM MgCl ₂ , 0.5 mM DTT, 0.05% Triton X-100, 5% glycerol, 1% BSA) and Incubated for 2 hours at 4 ° C. The array was then washed 3 times with probing buffer (1 minute on each wash on ice) and then incubated with Cy5-labeled goat anti-human IgG (0.5 μg / ml) at 4 ° C. for 1 hour. They were washed again as described above, dried by centrifugation (800 × g), set in a Cy5 filter and scanned in a Perkin-Elmer Proscan microarray scanner with stationary PMT.

  Epitope mapping by ortholog substitution was also performed. Since the tested antibodies recognize conformational or non-contiguous epitopes, it is only possible to determine each epitope in the context of a properly folded GM-CSF. As shown in the Biacore results described above, the antibody in question binds only weakly, if any, to mouse GM-CSF. Thus, the inventors designed a chimeric version of GM-CSF in which human GM-CSF in a selected region was replaced by the corresponding mouse sequence. Since human and mouse GM-CSF have only a 73% identity but probably a similar three-dimensional structure, chimeric proteins can be affected by alanine scanning methods (with polyalanine extension) that can affect protein folding. In contrast, the GM-CSF folding should be retained. A chimera in which the epitope or portion thereof has been replaced with a murine sequence should no longer bind to the antibody in question (or should show reduced binding). Returning the mapping of the sequence on the known human GM-CSF three-dimensional structure allows reconfiguration of the spatial position of the amino acids containing the epitope. Amino acids conserved in GM-CSF between humans and mice cannot be analyzed by this method.

  Eleven different chimeras were designed in which seven linear peptide sequences were replaced individually and in combination. For each chimera, mouse GM-CSF amino acids are shown in bold. Probable N-linked glycosylation sites that differ between chimeras are underlined. Each chimera was expressed in HEK293 cells as a rabbit Fc fusion protein that allows purification and quantification by the rabbit Fc portion. A Western blot of a control with a fusion protein using an antibody against rabbit Fc showed that the same amount of various GM-CSF chimeric fusion proteins were loaded on the gel. The shift in mobility between the various chimeras is due to differences in glycosylation between human and mouse GM-CSF.

  Western blots of GM-CSF chimera with EV1003 show that chimeras 3, 6, 9, and 10 show little or no binding. Chimera 9 is a combination of chimeras 3 and 6, and chimera 10 is a combination of chimeras 2 and 6. Thus, all chimeras containing murine amino acids at the positions represented by chimeras 3 and 6 or combinations comprising one or both of these regions are no longer recognized by EV1003. They therefore represent the EV1003 epitope. Spatial mapping of chimeras 3 and 6 onto the three-dimensional crystal structure of GM-CSF showed that these chimeras were adjacent to each other on one side of the GM-CSF molecule, Match. This surface represents a discontinuous epitope recognized by EV1003.

Western blots of GM-CSF chimera with EV1019 show that chimeras 4, 5, and 11 show weak binding and chimera 8 shows little binding. Chimera 11 is a combination of chimeras 1 and 5, and chimera 8 is a combination of chimeras 4 and 5. Thus, all chimeras containing mouse amino acids in the position represented by chimera 4 or 5 show weak binding to EV1019. Chimera 8 containing a combination of murine amino acids at the positions represented by chimeras 4 and 5 is no longer recognized by EV1019. Thus, the combination of chimeras 4 and 5 represents the EV1019 epitope. Spatial mapping of chimeras 4 and 5 onto the three-dimensional crystal structure of GM-CSF showed that these chimeras were adjacent to each other on one side of the GM-CSF molecule, Match. This surface represents a discontinuous epitope recognized by EV1019. Since both regions are required for high affinity binding of EV1019, the discontinuous epitopes are in close proximity to each other and are on or near the surface of GM-CSF (and thus exposed to the surface). Must consist of amino acids from variants 4 and 5 (which affect the amino acid residues present). These are amino acids 60-63 (ELYK) and 78-87 (TMMASHYKQH) (SEQ ID NO: 3), respectively. A contribution from amino acid 76 (P) may be possible. Due to the large distance from the interface between the involved chimeras 4 and 5, amino acids 68-69 (SL) in chimera 4 may not play an important role in the binding epitope, but this is completely excluded Can not do it.
Western blots with EV1018 of the GM-CSF chimera show that chimeras 4, 5, and 11 show weak binding and chimera 8 shows little binding. Chimera 11 is a combination of chimeras 1 and 5, and chimera 8 is a combination of chimeras 4 and 5. Thus, all chimeras containing mouse amino acids at the position represented by chimera 4 or 5 show weak binding to EV1018. Chimera 8 containing a combination of murine amino acids in the positions represented by chimeras 4 and 5 is no longer recognized by EV1018. Thus, the combination of chimeras 4 and 5 represents the EV1018 epitope. Spatial mapping of chimeras 4 and 5 onto the three-dimensional crystal structure of GM-CSF showed that these chimeras were adjacent to each other on one side of the GM-CSF molecule, Match. This surface represents a discontinuous epitope recognized by EV1018. Since both regions are required for high affinity binding of EV1018, the discontinuous epitopes are in close proximity to each other and are on or near the surface of GM-CSF (and thus exposed to the surface). Must consist of amino acids from variants 4 and 5 (which affect the amino acid residues present). These are the amino acids “ELYK” and “TMMASHYKQH”, respectively.

  These results show that EV1018 and EV1019 recognize the same discontinuous epitope of GM-CSF molecule, and EV1003 recognizes an epitope of GM-CSF molecule that is distinct from the epitope recognized by EV1018 and EV1019. Point out.

List of sequences full length hGM-CSF
MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE
(SEQ ID NO: 1)

Discontinuous epitope for EV1018 & EV1019
> Residues 77-80
ELYK
(SEQ ID NO: 2)

Residues 95-104
TMMASHYKQH
(SEQ ID NO: 3)

EV1018 CDRs
> EV1018 HC CDR1
SYGMH
(SEQ ID NO: 4)

> EV1018 HC CDR2
LTYHHGNRKFYADSVRG
(SEQ ID NO: 5)

> EV1018 HC CDR3
ESMGAINDN
(SEQ ID NO: 6)

> EV1018 LC CDR1
IGNNNNIGSHAVG
(SEQ ID NO: 7)

> EV1018 LC CDR2
GRSPPS
(SEQ ID NO: 8)

> EV1018 LC CDR3
STWDSSLSAVV
(SEQ ID NO: 9)

Heavy chain without EV1018 signal sequence
> EV1018
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 10)

> EV1018-wt IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 11)

> EV1018-T97A IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN A LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 12)

> EV1018-T97V IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN V LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 13)

> EV1018-N95D IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS D NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 14)

> EV1018-N95E IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS E NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 15)

> EV1018-N95K IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS K NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 16)

> EV1018-N95Q IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS Q NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 17)

> EV1018-N93Q-N95T IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRD Q S T NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 18)

> EV1018-wt IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 19)

> EV1018-T97A IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN A LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 20)

> EV1018-T97V IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN V LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 21)

> EV1018-N95D IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS D NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 22)

> EV1018-N95E IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS E NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 23)

> EV1018-N95K IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS K NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 24)

> EV1018-N95Q IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS Q NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 25)

> EV1018-N93Q-N95T IgG1-BI QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRD Q S T NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 26)

> EV1018-T97A IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN A LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 27)

> EV1018-T97V IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN V LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 28)

> EV1018-N95D IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS D NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 29)

> EV1018-N95E IgG1-original-stationary QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS E NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 30)

> EV1018-N95K IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS K NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 31)

> EV1018-N95Q IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS Q NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 32)

> EV1018-N93Q-N95T IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRD Q S T NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 33)

Light chain without EV1018 signal sequence
> EV1018-wt-original
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGKSTTTPSKQSLTQVTSGS
(SEQ ID NO: 34)

> EV1018-wt-BI
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGKSTTTPSKQSPEKY
(SEQ ID NO: 35)

> EV1018-wt-BI-G1
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGKSTTTPSKQSPE
(SEQ ID NO: 36)

> EV1018-wt-original-steady
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGKSTTTPSKQSLTQVTSGS
(SEQ ID NO: 37)

Heavy chain without EV1018 signal sequence
> EV1018-wt-IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 38)

> EV1018-T97A-IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 39)

> EV1018-T97V-IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 40)

> EV1018-N95D-IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 41)

> EV1018-N95E IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 42)

> EV1018-N95K IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 43)

> EV1018-N95Q IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 44)

> EV1018-N93Q-N95T IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 45)

> EV1018-wt IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 46)

> EV1018-T97A IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 47)

> EV1018-T97V IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 48)

> EV1018-N95D IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 49)

> EV1018-N95E IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 50)

> EV1018-N95K IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 51)

> EV1018-N95Q IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 52)

> EV1018-N93Q-N95T IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 53)

> EV1018-IgG2
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 54)

> EV1018-wt-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 55)

> EV1018-T97A-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 56)

> EV1018-T97V-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 57)

> EV1018-N95D-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 58)

> EV1018-N95E-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 59)

> EV1018-N95K-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 60)

> EV1018-N95Q-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 61)

> EV1018-N93Q-N95T-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 62)

> EV1018-IgG4
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 63)

> EV1018-wt-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 64)

> EV1018-T97A-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 65)

> EV1018-T97V-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 66)

> EV1018-N95D-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 67)

> EV1018-N95E-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 68)

> EV1018-N95K-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 69)

> EV1018-N95Q-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 70)

> EV1018-N93Q-N95T-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 71)

> EV1018-IgG4-SP
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 72)

> EV1018-wt-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 73)

> EV1018-T97A-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 74)

> EV1018-T97V-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 75)

> EV1018-N95D-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 76)

> EV1018-N95E-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 77)

> EV1018-N95K-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 78)

> EV1018-N95Q-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 79)

> EV1018-N93Q-N95T-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 80)

Heavy chain with EV1018 signal sequence
> EV1018
MEFGLIWVFLVTLLRGVQC QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 81)

> EV1018-wt-IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 82)

> EV1018-T97A-IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 83)

> EV1018-T97V-IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 84)

> EV1018-N95D-IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 85)

> EV1018-N95E IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 86)

> EV1018-N95K IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 87)

> EV1018-N95Q IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 88)

> EV1018-N93Q-N95T IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 89)

> EV1018-wt IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 90)

> EV1018-T97A IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 91)

> EV1018-T97V IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 92)

> EV1018-N95D IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 93)

> EV1018-N95E IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 94)

> EV1018-N95K IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 95)

> EV1018-N95Q IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 96)

> EV1018-N93Q-N95T IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 97)

> EV1018-T97A IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 98)

> EV1018-T97V IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 99)

> EV1018-N95D IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 100)

> EV1018-N95E IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 101)

> EV1018-N95K IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 102)

> EV1018-N95Q IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 103)

> EV1018-N93Q-N95T IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 104)

Light chain with EV1018 signal sequence
> EV1018-wt-original
MAWTPLLLQLLTLCSGSWA QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQSQATPEVQQQQQQPE
(SEQ ID NO: 105)

> EV1018-wt-BI
MGWSCIILFLVATATGVHS QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQASKATFPCLISEQSQQPE
(SEQ ID NO: 106)

> EV1018-wt-BI2 (G1) MGWSCIILFLVATATGVHS QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYDSSTSSLSSLSAVVFGGGTKLKPSQP
(SEQ ID NO: 107)

> EV1018-wt-original-steady
MGWSCIILFLVATATGVHS QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQSQQVPE
(SEQ ID NO: 108)

Heavy chain with EV1018 signal sequence
> EV1018-wt-IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 109)

> EV1018-T97A-IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 110)

> EV1018-T97V-IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 111)

> EV1018-N95D-IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 112)

> EV1018-N95E IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 113)

> EV1018-N95K IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 114)

> EV1018-N95Q IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 115)

> EV1018-N93Q-N95T IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 116)

> EV1018-wt IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 117)

> EV1018-T97A IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 118)

> EV1018-T97V IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 119)

> EV1018-N95D IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 120)

> EV1018-N95E IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 121)

> EV1018-N95K IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 122)

> EV1018-N95Q IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 123)

> EV1018-N93Q-N95T IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 124)

> EV1018-IgG2
MEFGLIWVFLVTLLRGVQC QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 125)

> EV1018-wt-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 126)

> EV1018-T97A-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 127)

> EV1018-T97V-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 128)

> EV1018-N95D-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 129)

> EV1018-N95E-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 130)

> EV1018-N95K-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 131)

> EV1018-N95Q-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 132)

> EV1018-N93Q-N95T-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 133)

> EV1018-IgG4
MEFGLIWVFLVTLLRGVQC QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 134)

> EV1018-wt-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 135)

> EV1018-T97A-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 136)

> EV1018-T97V-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 137)

> EV1018-N95D-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 138)

> EV1018-N95E-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 139)

> EV1018-N95K-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 140)

> EV1018-N95Q-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 141)

> EV1018-N93Q-N95T-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 142)

> EV1018-IgG4-SP
MEFGLIWVFLVTLLRGVQC QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 143)

> EV1018-wt-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 144)

> EV1018-T97A-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 145)

> EV1018-T97V-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 146)

> EV1018-N95D-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 147)

> EV1018-N95E-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 148)

> EV1018-N95K-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 149)

> EV1018-N95Q-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 150)

> EV1018-N93Q-N95T-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 151)

Heavy chain variable region without EV1019 signal sequence
> EV1019-VH-wt
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSS
(SEQ ID NO: 152)

> EV1019-VH-C105G
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSS
(SEQ ID NO: 153)

> EV1019-VH-C105S
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSS
(SEQ ID NO: 154)

> EV1019-VH-C105A
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSS
(SEQ ID NO: 155)

> EV1019-VH-C105T
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSS
(SEQ ID NO: 156)

> EV1019-VH-C105M
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSS
(SEQ ID NO: 157)

> EV1019-VH-C105Q
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSS
(SEQ ID NO: 158)

> EV1019-VH-C105L
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSS
(SEQ ID NO: 159)

Heavy chain without EV1019 signal sequence
> EV1019
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 160)

> EV1019-wt-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 161)

> EV1019-wt-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 162)

> EV1019-C105G
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 163)

> EV1019-C105G-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 164)

> EV1019-C105G-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 165)

> EV1019-C105S
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 166)

> EV1019-C105S-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 167)

> EV1019-C105S-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 168)

> EV1019-C105A
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 169)

> EV1019-C105A-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 170)

> EV1019-C105A-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 171)

> EV1019-C105Q
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 172)

> EV1019-C105Q-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 173)

> EV1019-C105Q-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 174)

> EV1019-C105T
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 175)

> EV1019-C105T-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 176)

> EV1019-C105T-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 177)

> EV1019-C105M
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 178)

> EV1019-C105M-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 179)

> EV1019-C105M-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 180)

> EV1019-C105L
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 181)

> EV1019-C105L-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 182)

> EV1019-C105L-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 183)

Light chain variable region without EV1019 signal sequence
> EV1019-VL-wt
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 184)

> EV1019-VL-BI
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 185)

> EV1019-VL-N25S
QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 186)

> EV1019-VL-BI-N25S
QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 187)

> EV1019-VL-N25G
QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 188)

> EV1019-VL-BI-N25G
QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 189)

> EV1019-VL-N25T
QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 190)

> EV1019-VL-BI-N25T
QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 191)

> EV1019-VL-N25R
QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 192)

> EV1019-VL-BI-N25R
QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 193)

> EV1019-VL-N25Q
QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 194)

> EV1019-VL-BI-N25Q
QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 195)

> EV1019-VL-S27N
QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 196)

> EV1019-VL-BI-S27N
QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 197)

> EV1019-VL-S27G
QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 198)

> EV1019-VL-BI-S27G
QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 199)

> EV1019-VL-S27A
QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLR
(SEQ ID NO: 200)

> EV1019-VL-BI-S27A
QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLG
(SEQ ID NO: 201)

EV1019 light chain without signal sequence
> EV1019-wt-original
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 202)

> EV1019-wt-BI
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSPEKYQTEGSTV
(SEQ ID NO: 203)

> EV1019-wt-BI2
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGKETSHR
(SEQ ID NO: 204)

> EV1019-wt-original-steady
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 205)

> EV1019-N25S
QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 206)

> EV1019-N25S-BI2
QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 207)

> EV1019-N25G
QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 208)

> EV1019-N25G-BI2
QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQ
(SEQ ID NO: 209)

> EV1019-N25T
QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 210)

> EV1019-N25T-BI2
QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 211)

> EV1019-N25R
QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGKETSHR
(SEQ ID NO: 212)

> EV1019-N25R-BI2
QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGKETSHRSKSYS
(SEQ ID NO: 213)

> EV1019-N25Q
QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 214)

> EV1019-N25Q-BI2
QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSPEKYQTEGSTV
(SEQ ID NO: 215)

> EV1019-S27N
QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGKETSHRSKLTPE
(SEQ ID NO: 216)

> EV1019-S27N-BI2
QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 217)

> EV1019-S27G
QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 218)

> EV1019-S27G-BI2
QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVEKSHRTPSYSLTPE
(SEQ ID NO: 219)

> EV1019-S27A
QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGEKTSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 220)

> EV1019-S27A-BI2
QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETSTPSKQSPEKYQTEGSTV
(SEQ ID NO: 221)

Heavy chain without EV1019 signal sequence
> EV1019-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 222)

> EV1019-wt-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 223)

> EV1019-wt-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 224)

> EV1019-C105G-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 225)

> EV1019-C105G-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 226)

> EV1019-C105G-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 227)

> EV1019-C105S-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 228)

> EV1019-C105S-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 229)

> EV1019-C105S-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 230)

> EV1019-C105A-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 231)

> EV1019-C105A-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 232)

> EV1019-C105A-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 233)

> EV1019-C105Q-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 234)

> EV1019-C105Q-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 235)

> EV1019-C105Q-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 236)

> EV1019-C105T-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 237)

> EV1019-C105T-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 238)

> EV1019-C105T-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 239)

> EV1019-C105M-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 240)

> EV1019-C105M-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 241)

> EV1019-C105M-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 242)

> EV1019-C105L-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 243)

> EV1019-C105L-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 244)

> EV1019-C105L-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 245)

Heavy chain with EV1019 signal sequence
> EV1019
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 246)

> EV1019-wt-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 247)

> EV1019-wt-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 248)

> EV1019-C105G
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 249)

> EV1019-C105G-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 250)

> EV1019-C105G-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 251)

> EV1019-C105S
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 252)

> EV1019-C105S-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 253)

> EV1019-C105S-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 254)

> EV1019-C105A
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 255)

> EV1019-C105A-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 256)

> EV1019-C105A-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 257)

> EV1019-C105Q
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 258)

> EV1019-C105Q-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 259)

> EV1019-C105Q-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 260)

> EV1019-C105T
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 261)

> EV1019-C105T-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 262)

> EV1019-C105T-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 263)

> EV1019-C105M
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 264)

> EV1019-C105M-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 265)

> EV1019-C105M-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 266)

> EV1019-C105L
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 267)

> EV1019-C105L-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 268)

> EV1019-C105L-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 269)

Light chain with EV1019 signal sequence
> EV1019-wt-original
MAWTPLLLQLLTLCSGSWA QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGATVPEWTT
(SEQ ID NO: 270)

> EV1019-wt-BI
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQVTKYWTT
(SEQ ID NO: 271)

> EV1019-wt-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVKYWTT
(SEQ ID NO: 272)

> EV1019-wt-original-steady
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVKYWTT
(SEQ ID NO: 273)

> EV1019-N25S
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVNWTT
(SEQ ID NO: 274)

> EV1019-N25S-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVNWTT
(SEQ ID NO: 275)

> EV1019-N25G
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVKYWTT
(SEQ ID NO: 276)

> EV1019-N25G-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVNWTT
(SEQ ID NO: 277)

> EV1019-N25T
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGATVKYWTT
(SEQ ID NO: 278)

> EV1019-N25T-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVNWTT
(SEQ ID NO: 279)

> EV1019-N25R
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTK L
(SEQ ID NO: 280)

> EV1019-N25R-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVNWTT
(SEQ ID NO: 281)

> EV1019-N25Q
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTK L
(SEQ ID NO: 282)

> EV1019-N25Q-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVKYWTT
(SEQ ID NO: 283)

> EV1019-S27N
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTK L
(SEQ ID NO: 284)

> EV1019-S27N-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVKYWTT
(SEQ ID NO: 285)

> EV1019-S27G
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVNWTT
(SEQ ID NO: 286)

> EV1019-S27G-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVNWTT
(SEQ ID NO: 287)

> EV1019-S27A
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVKYWTT
(SEQ ID NO: 288)

> EV1019-S27A-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPQTVKYWTT
(SEQ ID NO: 289)

Heavy chain with EV1019 signal sequence
> EV1019-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 290)

> EV1019-wt-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 291)

> EV1019-wt-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 292)

> EV1019-C105G-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 293)

> EV1019-C105G-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 294)

> EV1019-C105G-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 295)

> EV1019-C105S-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 296)

> EV1019-C105S-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 297)

> EV1019-C105S-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 298)

> EV1019-C105A-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 299)

> EV1019-C105A-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 300)

> EV1019-C105A-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 301)

> EV1019-C105Q-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 302)

> EV1019-C105Q-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 303)

> EV1019-C105Q-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 304)

> EV1019-C105T-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 305)

> EV1019-C105T-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 306)

> EV1019-C105T-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 307)

> EV1019-C105M-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 308)

> EV1019-C105M-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 309)

> EV1019-C105M-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 310)

> EV1019-C105L-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 311)

> EV1019-C105L-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 312)

> EV1019-C105L-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 313)

EV1018 and EV1019 conserved sequences
> V _H -CDR1
FTFSX ₁ X ₂ MH
X ₁ is Y or H, and X ₂ is G or A
(SEQ ID NO: 314)

> V _H -CDR2
X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G
Amino each X _n is any naturally occurring independently, X ₃ is L or V, X ₄ is T or I, X ₅ is Y or W, X ₆ is R or K, X ₇ is F or Y, and X ₈ is R or K
(SEQ ID NO: 315)

> V _H -CDR3
EX _n X ₉ GX ₁₀ X _n X _n DX _n
Each X _n is independently any naturally occurring amino acid, X ₉ is M or V, and X ₁₀ is A or G
(SEQ ID NO: 316)

> V _L -CDR1
X _n GNX _n X _n NIGSX ₁₁ AVG
Each X _n is independently any naturally occurring amino acid, and X ₁₁ is H or Y
(SEQ ID NO: 317)

> V _L -CDR2
GX ₁₂ SPX ₁₃ SG
X ₁₂ is R or K, and X ₁₃ is A or P
(SEQ ID NO: 318)

> V _L -CDR3
STWDSX ₁₄ LSAVX ₁₅
X ₁₄ is R or S, and X ₁₅ is V or L
(SEQ ID NO: 319)

EV1003 and EV1007 array
> EV1003 heavy chain (IgG1)
MDWTWRILFLVAAATGARSLVQLVQSEAEVKKPGASVRISCRTSGYIFPTFALHWVRQAPGQSLEWVGSINTASGKTKFSTKFQDRLTLSSNTSATTVYMDLSGLTLDDTALYYCARDRFQNIMATILDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 320)

> EV1003 light chain (κ)
METPAQLLFLLLLWLPGTTGDIVLTQSPGTLSLSPGERVTVSCRASHRVSSNYLAWYQQRPGQSPRLLIFGASNRATGVPDRFSGSGSGTDFALTISRLEPDDFAVYYCQQYASSPVTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNKVTYTEQS
(SEQ ID NO: 321)

> EV1007 heavy chain (IgG1)
MDWTWRILFLVAAATGAPSQVHLVQSGSELKKPGASVKVSCKASGYSFSRYGIKWVRQAPGQGLEWMGWINTRSGVPAYAQGFTGRFVFSLDTSVDTAFLEISSLKTEDTGIYYCATRPPRFYDKTEYWEDGFDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK
(SEQ ID NO: 322)

> EV1007 light chain (λ)
MAGFPLLLTLLTHCAGSWAQSVLTQPPSASGTPGQSVNISCSGSSSNIGNSYVYWYQQLPGTAPKLLIYRNNRRPSGVPDRFSGSKSDTSASLAISGLRSEDEADYYCATWDDSLSGRLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGATVVAWTT
(SEQ ID NO: 323)

Signal sequence and further sequences
> EV1018
MEFGLIWVFLVTLLRGVQC
(SEQ ID NO: 324)

> EV1018-wt IgG1KO
MGWSCIILFLVATATGVHS
(SEQ ID NO: 325)

> EV1018 wt-original
MAWTPLLLQLLTLCSGSWA
(SEQ ID NO: 326)

TGTSSDVENYNLVS
(SEQ ID NO: 327)

EGSKRPS
(SEQ ID NO: 328)

CSYAGSSVVV
(SEQ ID NO: 329)

SGNSSNIGSYAVG
(SEQ ID NO: 330)

GKSPAS
(SEQ ID NO: 331)

STWDSRLSAVL
(SEQ ID NO: 332)

SHAMH
(SEQ ID NO: 333)

VIWHDGSKKYYADSVKG
(SEQ ID NO: 334)

EWVGGTCDS
(SEQ ID NO: 335)

SGSSSNIGNSYVY
(SEQ ID NO: 336)

RNNRRPS
(SEQ ID NO: 337)

ATWDDSLSGRL
(SEQ ID NO: 338)

RYGIK
(SEQ ID NO: 339)

WINTRSGVPAYAQGFTG
(SEQ ID NO: 340)

RPPRFYDKTEYWEDGFDV
(SEQ ID NO: 341)

RASHRVSSNYLA
(SEQ ID NO: 342)

GASNRAT
(SEQ ID NO: 343)

QQYASSPVT
(SEQ ID NO: 344)

TFALH
(SEQ ID NO: 345)

SINTASGKTKFSTKFQD
(SEQ ID NO: 346)

DRFQNIMATILDV
(SEQ ID NO: 347)

EV1018 heavy chain variable region
> EV1018-VH
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 348)

> EV1018-VH-wt
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 349)

> EV1018-VH-T97A
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN A LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 350)

> EV1018-VH-T97V
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN V LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 351)

> EV1018-VH-N95D
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS D NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 352)

> EV1018-VH-N95E
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS E NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 353)

> EV1018-VH-N95K
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS K NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 354)

> EV1018-VH-N95Q
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS Q NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 355)

> EV1018-VH-N93Q-N95T
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRD Q S T NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 356)

> EV1018-VH-T97A IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN A LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 357)

> EV1018-VH-T97V IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN V LYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 358)

> EV1018-VH-N95D IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS D NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 359)

> EV1018-VH-N95E IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS E NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 360)

> EV1018-VH-N95K IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS K NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 361)

> EV1018-VH-N95Q IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS Q NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 362)

> EV1018-VH-N93Q-N95T IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRD Q S T NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 363)

EV1018 light chain variable region
> EV1018-VL-wt-original
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLR
(SEQ ID NO: 364)

> EV1018-VL-wt-BI
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLG
(SEQ ID NO: 365)

Claims (23)

(A) a heavy chain comprising a VH-CDR1 containing sequence, a VH-CDR2 containing sequence and a VH-CDR3 containing sequence,
(I) the VH-CDR1-containing sequence is SYGMH (SEQ ID NO: 4);
(Ii) the VH-CDR2 containing sequence is LTYHHGNRKFYADSVRG (SEQ ID NO: 5);
(Iii) the heavy chain wherein the VH-CDR3-containing sequence is ESMGAINDN (SEQ ID NO: 6); and
(B) a light chain comprising a VL-CDR1 containing sequence, a VL-CDR2 containing sequence and a VL-CDR3 containing sequence,
(I) the VL-CDR1-containing sequence is IGNNNNIGSHAVG (SEQ ID NO: 7);
(Ii) the VL-CDR2 containing sequence is GRSPPS (SEQ ID NO: 8);
(Iii) the light chain, wherein the VL-CDR3 containing sequence is STWDSSLSAVV (SEQ ID NO: 9);
An isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof.   2. The antibody or antigen-binding fragment thereof according to claim 1, which binds to human GM-CSF with a KD of less than 400 pM.   The antibody or antigen-binding fragment thereof according to claim 1 or 2, which neutralizes hGM-CSF activity, as measured by ED80 in a TF-1 proliferation assay, wherein the antibody or antigen-binding fragment thereof has an IC50 value of less than 100 pM.   The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain is selected from the group consisting of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3) and gamma 4 (γ4).   The antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain is gamma 1 (γ1).   The antibody or antigen-binding fragment thereof according to claim 4 or 5, wherein the light chain is a lambda light chain.   The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain has the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 51.   The antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain has the amino acid sequence of SEQ ID NO: 51.   The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the light chain has the amino acid sequence of SEQ ID NO: 36. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain variable region sequence is SEQ ID NO: 348 or SEQ ID NO: 361, and the light chain variable region sequence is SEQ ID NO: 365.   The antibody or antigen-binding fragment thereof according to claim 10, wherein the heavy chain variable region sequence is SEQ ID NO: 361.   The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain sequence is SEQ ID NO: 46 or SEQ ID NO: 51, and the light chain sequence is SEQ ID NO: 36.   The antibody or antigen-binding fragment thereof according to claim 12, wherein the heavy chain sequence is SEQ ID NO: 51.   A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier. (A) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, and
(B) A kit comprising one or more containers containing the antibody or antigen-binding fragment thereof.   An isolated nucleic acid encoding the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-13.   The isolated nucleic acid of claim 16 which is DNA.   A vector comprising the DNA according to claim 17.   19. A host cell comprising the vector of claim 18, wherein the vector is an expression vector.   A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1 for the treatment of a disease or disorder associated with overexpression of hGM-CSF in a subject.   21. The pharmaceutical composition of claim 20, wherein the antibody or antigen-binding fragment is administered to the subject in an amount not exceeding 500 mg per dose.   The disease or disorder is chronic obstructive pulmonary disease (COPD), asthma, bronchial asthma, childhood asthma, severe asthma, acute asthma attack, cystic fibrosis, interstitial lung disease, rhinitis, arthritis and related joint diseases, rheumatoid arthritis The pharmaceutical composition according to claim 20 or 21, selected from the group consisting of: psoriasis, myeloid leukemia and multiple sclerosis. The method of producing an anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof according to claim 1 that binds to hGM-CSF, wherein the antibody or antigen-binding fragment thereof is at least a VH-CDR1-containing sequence, a VH-CDR2-containing sequence. , A VH-CDR3 containing sequence, a VL-CDR1 containing sequence, a VL-CDR2 containing sequence and a VL-CDR3 containing sequence,
(I) at least one encoding at least the VH-CDR1-containing sequence, the VH-CDR2-containing sequence, the VH-CDR3-containing sequence, the VL-CDR1-containing sequence, the VL-CDR2-containing sequence, and the VL-CDR3-containing sequence. Obtaining a host cell comprising a DNA sequence of a species, comprising:
(A) the VH-CDR1-containing sequence is SYGMH (SEQ ID NO: 4);
(B) the VH-CDR2 containing sequence is LTYHHGNRKFYADSVRG (SEQ ID NO: 5);
(C) the VH-CDR3-containing sequence is ESMGAINDN (SEQ ID NO: 6),
(D) the VL-CDR1-containing sequence is IGNNNNIGSHAVG (SEQ ID NO: 7);
(E) the VL-CDR2 containing sequence is GRSPPS (SEQ ID NO: 8);
(F) the VL-CDR3-containing sequence is STWDDSLSAVV (SEQ ID NO: 9), and (ii) culturing the host cell under conditions suitable for expression of DNA and production of an antibody or antigen-binding fragment thereof,
Said method.

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