JP6014706B2 - Monoclonal antibody binding to hGM-CSF and pharmaceutical composition comprising said antibody - Google Patents

Description

The present invention provides 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, Enhances 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, G
M-CSF is considered to be 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 h
It binds to GM-CSF and inhibits its physiological activity. This is, for example, an anti-hGM that has high affinity, has sufficient neutralizing activity for hGM-CSF, but does not induce an immunological response.
-Can be performed by administering a CSF monoclonal antibody.

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 inventor's development 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.

The present invention provides the following.
[1] An isolated anti-hGM-CSF monoclonal antibody or an antigen-binding fragment thereof that binds to an epitope of hGM-CSF (SEQ ID NO: 1) including ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3).
[2] binds with a K _D of less than 400pM human GM-CSF, the [1] an antibody or antigen-binding fragment thereof according.
[3] The antibody according to [1] or [2] above, wherein the antibody or antigen-binding fragment thereof has an IC50 value of less than 100 pM and neutralizes hGM-CSF activity as measured by ED80 in a TF-1 proliferation assay Or an antigen-binding fragment thereof.
[4] The above [1] or [2] , wherein the heavy chain is selected from the group consisting of gamma 1 (γ ₁ ), gamma 2 (γ ₂ ), gamma 3 (γ ₃ ) and gamma 4 (γ ₄ ). Or an antigen-binding fragment thereof.
[5] The antibody or antigen-binding fragment thereof according to [4] above, wherein the heavy chain is gamma 1 (γ ₁ ).
[6] The antibody or antigen-binding fragment thereof according to [4] or [5] above, wherein the light chain is a lambda light chain.
[7] (a) a heavy chain comprising a V _H -CDR1 containing sequence, a V _H -CDR2 containing sequence and a V _H -CDR3 containing sequence,
(I) the V _H -CDR1-containing sequence is SHAMH (SEQ ID NO: 333);
(Ii) The V _H -CDR2 containing sequence is VIWHDGSKKYYADSVKG (SEQ ID NO: 334),
(Iii) the heavy chain, wherein the V _H -CDR3-containing sequence is EWGVGTCDS (SEQ ID NO: 335);
and,
(B) (b) V _L -CDR1 containing sequence, V _L -CDR2 containing sequence and V _L -CDR3
A light chain comprising a containing sequence,
(I) the V _L -CDR1-containing sequence is SGNSSNIGSYAVG (SEQ ID NO: 330),
(Ii) the V _L -CDR2 containing sequence is GKSPAS (SEQ ID NO: 331);
(Iii) the light chain, wherein the V _L -CDR3-containing sequence is STWDSRLSAVL (SEQ ID NO: 332),
The antibody or antigen-binding fragment thereof according to any one of [1] to [6] above, comprising
[8] The antibody or antigen-binding fragment thereof according to any one of [1] to [7] above, wherein the heavy chain has the amino acid sequence of SEQ ID NO: 160.
[9] The antibody or antigen-binding fragment thereof according to any one of [1] to [8] above, wherein the heavy chain has the amino acid sequence of SEQ ID NO: 202.
[10] The antibody or antigen binding thereof according to any one of [1] to [7] above, wherein the heavy chain variable region sequence is SEQ ID NO: 152 and the light chain variable region sequence is SEQ ID NO: 184 fragment.
[11] The antibody or antigen-binding fragment thereof according to any one of [1] to [7] above, wherein the heavy chain sequence is SEQ ID NO: 160 and the light chain sequence is SEQ ID NO: 202.
[12] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [11] above and a pharmaceutically acceptable carrier.
[13] (a) The antibody or antigen-binding fragment thereof according to any one of [1] to [11] above, and (b) one or more containers containing the antibody or antigen-binding fragment thereof A kit comprising:
[14] An isolated nucleic acid encoding the anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [11] above.
[15] The isolated nucleic acid according to [14], which is DNA.
[16] A vector comprising the DNA of [15] above.
[17] A host cell comprising the vector according to [16] above, wherein the vector is an expression vector.
[18] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to [1] to [11] above for treating a disease or disorder associated with overexpression of hGM-CSF in a subject.
[19] The pharmaceutical composition of the above-mentioned [18], wherein the antibody or antigen-binding fragment is administered to the subject in an amount not exceeding 500 mg per dose.
[20] 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 The pharmaceutical composition according to [18] or [19] above, selected from the group consisting of Rheumatoid arthritis, psoriasis, myeloid leukemia and multiple sclerosis
[21] The anti-hGM-CSF monoclonal antibody according to [1], which binds to hGM-CSF, or
A method for producing the antigen-binding fragment, wherein the antibody or the antigen-binding fragment thereof is at least a V _H -CDR1-containing sequence, a V _H -CDR2 containing sequence, a V _H -CDR3 containing sequence, a V _L -CDR1 containing sequence, V Comprising an _L- CDR2 containing sequence and a V _L -CDR3 containing sequence,
(A) at least the V _H -CDR1 containing sequence, the V _H -CDR2 containing sequence, the V _H -CDR3 containing sequence, the V _L -CDR1 containing sequence, the V _L -CDR2 containing sequence and the V _L -CDR3 containing Obtaining a host cell comprising at least one DNA sequence encoding the sequence, comprising:
(I) the V _H -CDR1-containing sequence is SHAMH (SEQ ID NO: 333);
(Ii) The V _H -CDR2 containing sequence is VIWHDGSKKYYADSVKG (SEQ ID NO: 334),
(Iii) the V _H -CDR3 containing sequence is EWGVGTCDS (SEQ ID NO: 335),
(Iv) the V _L -CDR1-containing sequence is SGNSSNIGSYAVG (SEQ ID NO: 330),
(V) the V _L -CDR2 containing sequence is GKSPAS (SEQ ID NO: 331),
(Vi) The _VL- CDR3-containing sequence is STWDSRLSAVL (SEQ ID NO: 332)
Being, and
(B) The method comprising culturing the host cell under conditions suitable for expression of DNA and production of an antibody or antigen-binding fragment thereof.
[22] The pharmaceutical composition according to any one of the above [18] to [20], further comprising a pharmaceutically acceptable carrier.

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 is SYGMH (SEQ ID NO: 10) and SHAMH (SEQ ID NO: 3)
33) having an amino acid sequence selected from
(Ii) V _H -CDR2 has an amino acid sequence selected from LTYHHGNRKFYADSVRG (SEQ ID NO: 5) and VIWHGDSKKYYADSVKG (SEQ ID NO: 334);
(Iii) V _H -CDR3 is ESMGAINDN (SEQ ID NO: 6) and EWVGGT
A heavy chain having an amino acid sequence selected from CDS (SEQ ID NO: 335), and (b) a light chain comprising V _L -CDR1, V _L -CDR2 and V _L -CDR3,
(I) V _L -CDR1 is IGNNNNIGSHAVG (SEQ ID NO: 7) and SGNS
Having an amino acid sequence selected from SNIGSYAVG (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 is STWDDSLSAVV (SEQ ID NO: 9) and STWD
A light chain having an amino acid sequence selected from SRLSAVL (SEQ ID NO: 332), wherein the antibody or antigen-binding fragment thereof specifically binds to hGM-CSF.
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 <100 pM (eg <30 pM, <20 pM)
It is.

In any of the embodiments, the antibody or antigen-binding fragment thereof is gamma 1 (γ ₁ ),
Heavy chains that are gamma 2 (γ ₂ ), gamma 3 (γ ₃ ), or gamma 4 (γ ₄ ) can be included. Heavy chains are SEQ ID NOs: 10-33, 38-80 and 160-183 and 222-24
5 can be selected.
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
Compared to the wild type sequence, Q3E, T97A, T97V, N95D, N95E, N95K,
One or more amino acid substitutions selected from the group consisting of N95Q, N93Q / N95T, K144R, L164A and L165A can be included. In some embodiments, the antibody or antigen-binding fragment thereof is Q3E, T97A, T97V, N95D, N9.
5E, N95K, N95Q, N93Q / N95T, K144R, L164A and L16
A heavy chain containing one or more amino acid substitutions selected from the group consisting of 5A and a light chain 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 has the amino acid sequence LTYHHGNRKFY
ADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVKG (SEQ ID NO: 33)
_VH- CDR2 with 4) can be included. The antibody or antigen-binding fragment thereof described herein has the amino acid sequence ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS.
V _H -CDR3 having (SEQ ID NO: 335) can be included. 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 has the amino acid sequence GRSPPS.
(SEQ ID NO: 8) or V _L -CDR2 with GKSPAS (SEQ ID NO: 331) can be included. The antibody or antigen-binding fragment thereof described herein has the amino acid sequence STWDS
V _L -CDR3 with SLSAVV (SEQ ID NO: 9) or STWDSLSAVL (SEQ ID NO: 332) can be included.
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 recited herein, the antibody is a gamma 1 (γ ₁ ), gamma 2
It can comprise a heavy chain that is (γ ₂ ), gamma 3 (γ ₃ ) or gamma 4 (γ ₄ ) and a light chain that is kappa or lambda.
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 amino acid sequence is SEQ ID NO: 18.
4 or a light chain variable region that is a variant thereof selected from the group consisting of 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 amino acid sequence of the region and the light chain variable region is SEQ ID NO: 36.
4 or the light chain variable region of 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 is IgG ₁ (
[lambda] belongs to a 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. Anti-hGM as described herein
-CSF monoclonal antibody or antigen-binding portion thereof is expressed by TF-1 cells as hGM-CSF
The anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof may be characterized by inhibiting TF-1 cell proliferation by about 50% at a concentration of about 14 pM when proliferation is induced by. 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 invention further provides a host cell comprising an expression vector comprising the DNA vector of the 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 provides an anti-hGM-CSF according to the invention for use in medicine.
A monoclonal antibody or antigen-binding fragment thereof.
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 a plurality of types of anti-G, each as provided herein.
M-CSF antibodies, fragments or combinations thereof. For example, the composition further comprises hGM-CS
A second isolated antibody that binds to F, or an antigen-binding fragment thereof, and therefore the composition comprises a plurality of type / type (s) of antibodies, each of which binds to hGM-CSF, their Including an antigen-binding fragment, or a combination thereof. In some embodiments, hGM-CS
At least one of a plurality of antibodies that bind to F, an antigen-binding fragment thereof, or a combination thereof,
A polypeptide selected from the group consisting of SEQ ID NOs: 10-80, 152-245, 320-323, and 348-365.
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. hG
Examples of diseases caused by M-CSF overproduction 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) having a 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 hGM-C
An anti-hGM-CSF monoclonal antibody specific for SF or an 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:
Includes 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,
Selected from the group consisting of myeloid leukemia and 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. For such use, a composition comprising an antibody or antigen-binding fragment thereof or the same as described in the present invention,
Administration to a subject at a dose not exceeding the maximum tolerated dose, the maximum tolerated dose being about 500 mg per dose.
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. A composition comprising an antibody or antigen-binding fragment thereof is at least 1 in a subject
Administration in an amount effective to obtain a therapeutic effect of the species.
The present invention also includes 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 SF antibody-mediated hGM-CSF neutralizing activity is included. In some embodiments, the two or more antibodies, or antigen-binding portions thereof, are (a) and (b):
(A) having a 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 hGM-C
An anti-hGM-CSF monoclonal antibody specific for SF or an antigen-binding portion thereof, and (b) having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added, and hGM-CSF contains Specific (a) anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof is selected.

Embodiments show that two or more antibodies or antigen-binding portions thereof have 80% proliferation of TF-1 cells at a concentration of about 55 pM each when TF-1 cells are induced to grow by hGM-CSF.
A method for enhancing activity, which is characterized by the above inhibition, is included.
The invention also includes hGM- recognized by the antibodies and antigen-binding fragments described herein.
CSF epitopes are provided. 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). Containing groups 95-104, SEQ ID NOs: 4-9
Or anti-hGM-CSF comprising 6 different CDRs set forth in SEQ ID NOs: 330-335
Recognized by a monoclonal antibody or antigen-binding fragment thereof.
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. Molecules that bind to the epitope are, for example, anti-h
It may be a GM-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: 3
14), wherein X ₁ is Y or H, X ₂ is G or A,
(B) Consensus V _H -CDR2 containing sequence is X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADS
VX 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, and 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) 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.
, 152-183, 222-245, 348-362 and 363, wherein the light chain or part thereof is SEQ ID NO: 34-37, 184-221, 364 and 36
Having a sequence selected from the group consisting of 5;
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 is V _H -CDR1, V _H -CDR2 and V _H -C.
Including DNA encoding DR3,
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 VIWH
DGSKKYYADSVKG (SEQ ID NO: 334),
V _H -CDR3 is ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS (SEQ ID NO: 335).
In some embodiments, the vector is _VL- CDR1, _VL- CDR2, and _VL- CD.
Including DNA encoding R3,
V _L -CDR1 is IGNNNNIGSHAVG (SEQ ID NO: 7) or SGNNSNI
GYAVG (SEQ ID NO: 330),
V _L -CDR2 is either GRPSPPSG (SEQ ID NO: 8) or GKSPASG (SEQ ID NO: 3
31)
V _L -CDR3 is either STWDDSLSAVV (SEQ ID NO: 9) or STWDDSLLSA
VL (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 recombinant expression vector (s)
Host cells that express 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. FIG. 2 shows two graphs representing dose-dependent inhibition of rhesus monkey GM-CSF. FIG. 5 is a bar graph depicting myeloperoxidase (MPO) activity measured in bronchoalveolar fluid (BALF) pellets from mice exposed to cigarette smoke. CS: vehicle treated (300 μl vehicle ip on day 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: vehicle treated (300 μl vehicle ip on day 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 relates to a GM-CSF binding monoclonal antibody that exhibits improved neutralization of ability or activity against hGM-CSF and its G
M-CSF binding fragments and compositions comprising at least one or one type of such monoclonal antibodies or fragments thereof and methods of treating conditions or diseases associated with overexpression of hGM-CSF (for symptoms, diseases or disorders) Note that it provides higher hGM-CSF levels in individuals suffering from symptoms, diseases or disorders than in unaffected individuals.
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. Anti-hGM-CSF monoclonal antibody is an existing anti-hGM
It shows higher neutralizing activity (against hGM-CSF) than GM-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 invention allow the antigen's epitope to be dissociated throughout the antigen (eg, hGM) when the epitope of the antigen is observed in a natural conformation or structural study.
-CSF) are brought together in close proximity, thereby forming an epitope (eg, a “pocket” on the antigen surface) that is recognized together by the antibody of the present invention. It should be understood that the three-dimensional structure is recognized. 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 an antigen (eg, hG
M-CSF) represents any short variant of an antibody that exhibits specific antigen binding activity.
In particular, the fragments of the present invention have the same epitope as the respective monoclonal antibodies and hGM-CS.
A fragment that binds to F. 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. Therefore, “hGM-
“CSF bioactivity” 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 a patient with erythroleukemia. This assay is used for G of TF-1 cells.
Based on inhibition of M-CSF stimulated cell proliferation 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 was performed at various concentrations of wild type recombinant human GM-CSF (rhGM-C
In the presence of SF). 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,
Proliferation assay systems can be used to measure the neutralizing ability of antibodies to hGM-CSF. Other examples of assays for measuring biological GM-CSF function include I
Induction of L-8 secretion assay and surface CD11b / Mac1 assay are included.
Terms such as “neutralize”, “neutralize”, “neutralize” and the like refer to bioactive factors such as h
It means inhibition of the biological activity of GM-CSF. Therefore, an antibody against hGM-CSF with neutralizing ability or neutralizing activity is the same as that of hGM-CSF in biological systems.
And antagonize or counteract the biological activity of hGM-CSF, eg, the proliferative effect of hGM-CSF in biological systems such as TF-1 cells, thereby hGM-
It means that the biological activity of CSF can be substantially inhibited or eliminated.
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, the maximum half of the substance (5
0%) Inhibitory concentration (IC) (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 the neutralizing antibody against hGM-CSF produces a dose response curve and h
It can be determined by examining the effect of various concentrations of neutralizing antibody on reversal of the growth activity of GM-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, <50 pM, 60 pM
Less than, 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 40 pM as measured by ED80 in a TF-1 proliferation assay.
Is less than.

The anti-hGM-CSF monoclonal antibody in the present disclosure comprises an immunoglobulin molecule comprising two heavy chains and two light chains. Each heavy chain is represented by a heavy chain variable region (abbreviated appropriately as “HCVR” or “V _H ”) and a heavy chain constant region (heavy chain constant regions are “CH1”, “C
With 3 domains abbreviated as “H2” and “CH3”). 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 LCV
R is particularly important for the binding specificity of the antibody.
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 also have a relatively stable “framework region (FR)”
”And hypervariable“ complementarity determining regions (CDRs) ”. HCVR and LCVR
Each consists of 3 CDRs and 4 FRs. The order is FR1, CDR1, FR2, CDR2, FR3, CDR3 from the amino terminus to the carboxy terminus.
And FR4.

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 of the anti-hGM-CSF monoclonal antibodies or fragments thereof described herein
One type or kind 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). In addition, one or more types or types of anti-hGM
-CSF monoclonal antibodies or fragments thereof can be used or administered in combination with one or more other therapeutic agents that are not antibodies, such as small molecules, RNAi or peptides.

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
HGM-CSF that binds to and anti-hGM-CSF monoclonal antibody that binds to an antigen-binding fragment 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, anti-hGM-CSF monoclonal antibodies and antigen-binding fragments thereof that bind to hGM-CSF and exhibit neutralizing ability or activity against hGM-CSF are isolated,
This monoclonal antibody and antigen-binding fragment thereof recognize ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF (SEQ ID NO: 1). hGM
-Isolated anti-hGM-CSF monoclonal antibody and antigen-binding fragment thereof that bind to hGM-CSF recognizing ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in CSF are neutralizing against hGM-CSF activity Ability or activity, hGM-C
It is useful for treating and preventing diseases, conditions and disorders associated with SF overexpression. 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 comprises hGM-C
Amino acids at positions 77-80 in SF: ELYK (SEQ ID NO: 2) and hGM-CS
Recognizes the following defined hGM-CSF discontinuous epitope (SEQ ID NO: 1) corresponding to TMMASHYKQH (SEQ ID NO: 3) at positions 95-104 in F. The isolated anti-hGM-CSF monoclonal antibodies and antigen-binding fragments thereof described herein are hGM
-Specific binding to 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 EV1018, EV1019, as summarized in Tables 1 and 2 in the Examples.
, EV1003 and EV1007. Therefore, EV1018, EV1019
EV1003 and EV1007 can bind to hGM-CSF and neutralize the growth promoting effect of hGM-CSF. As shown in more detail, EV1018 and EV10
The 19 antibody recognizes the same or substantially overlapping epitope on the 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. An isolated DNA encoding an amino acid sequence comprising at least one selected from the group consisting of SEQ ID NOs: 4-9
Is provided.
One embodiment of the present invention binds to hGM-CSF (hGM-CSF) and hGM-CSF (
hGM-CSF) anti-hGM-CSF monoclonal antibody or antigen-binding portion, which can neutralize the biological activity, represented by at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 4-9 The present invention relates to an anti-hGM-CSF monoclonal antibody or antigen-binding portion characterized by having a complementary determining region (CDR).

Another embodiment of the present invention is an anti-hGM-CSF monoclonal antibody or antigen-binding moiety that can bind to hGM-CSF and neutralize the biological activity of hGM-CSF, comprising:
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: 330 to 335.
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 hGM-CSF
As long as it specifically binds to and neutralizes its biological activity.
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, and comprising the anti-hGM-C
The SF monoclonal antibody or 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 a hGM
-It is further incorporated into the present invention when it specifically binds to 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 An epitope of hGM-CSF, a discontinuous segment of human GM-CSF, is also the subject of this specification, which epitope comprises amino acid residues 77-
80 and human GM-CSF (SEQ ID NO: 1) amino acid residues 95-104, defined by the polypeptide sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3,
9 or anti-hGM-C comprising 6 different CDRs set forth in SEQ ID NOs: 330-335
Recognized by the SF monoclonal antibody or antigen-binding fragment thereof.
As described in more detail in the examples, monoclonal antibodies EV1018 and EV1
The epitope of hGM-CSF recognized by 019 was 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, EV1018 and EV101
Analysis of 9 CDR sequences 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;
For V _H -CDR2: X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 3
15), wherein each X _n is independently any natural amino acid, X ₃ is L or V, and 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;

For V _H -CDR3: EX _n X ₉ GX ₁₀ X _n X _n DX _n (SEQ ID NO: 316), where each X
_n is independently any natural amino acid, X ₉ is M or V, and 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 _1
4 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. Thus, another embodiment herein binds to hGM-CSF and hGM-CS
An isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof that exhibits neutralizing ability or activity against F, wherein the antibody or antigen-binding fragment thereof is (a) (i) X ₁
A consensus V _H -CDR1-containing sequence FTF in which Y is Y or H and X ₂ is G or A
SX 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 a consensus V _H -CD in which X ₆ is R or K, X ₇ is F or Y, and X ₈ is R or K.
R2-containing sequences 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, and X ₉ is M or V 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), wherein X ₁₀ is A or G, or a portion of the heavy chain in the case of an antibody fragment And (b) (i) a consensus V _L -C wherein each X _n is independently any natural amino acid and X ₁₁ is H or Y.
DR1-containing sequence _{X n GNX n X n NIGSX 11} AVG ( SEQ ID NO: 317), (ii) X ₁₂ is R or K, consensus V _L -CDR2 containing sequence GX ₁₂ X ₁₃ is A or P
SPX ₁₃ SG (SEQ ID NO: 318), and (iii) X ₁₄ is R or S, and X ₁₅ is V
Or a light chain comprising the consensus V _L -CDR3-containing sequence STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319), which is _L , or in the case of an antibody fragment, a portion of the light chain, which antibody or fragment thereof is ELYK in hGM-CSF (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) are recognized. 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-C that binds hGM-CSF.
SF monoclonal antibody or antigen-binding fragment thereof that binds hGM-CSF, the antibody or fragment thereof comprising (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, A consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n where 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 GX _n X ₆ KX ₇ YA
DSVX ₈ G (SEQ ID NO: 315), and (iii) Consensus V _H -CDR3 containing each X _n is independently any natural amino acid, X ₉ is M or V, and X ₁₀ is A or G A heavy chain comprising the sequence 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) each X _n is independently any Natural amino acid, X ₁
Consensus V _L -CDR1 containing sequence X ₁ G is a H or Y X _n GNX _n X _n NIGSX ₁₁ A
VG (SEQ ID NO: 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 (i
ii) 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 a portion of the light chain in the case of an antibody fragment Wherein the antibody or fragment thereof binds to hGM-CSF with a K _D of 400 pM or less, in another embodiment less than 160 pM, and the K _D is determined according to the technique 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 inhibits proliferation of peripheral blood dendritic cells. Thus, a further embodiment is an isolated anti-hGM-binding to hGM-CSF.
A CSF monoclonal antibody or antigen-binding fragment thereof that binds to hGM-CSF, the anti-hGM-CSF monoclonal antibody or fragment thereof comprising a heavy chain or a portion thereof and a light chain or a portion thereof. In the case of heavy chains or antibody fragments, a portion of the heavy chain is V _H −
CDR1, V _H -CDR2 and V _H -CDR3 can be included. 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, X ₆ is R or K, X ₇ Is F or Y and X ₈ is R
Or the formula X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G (SEQ ID NO: 315), which is or K, represents a consensus sequence comprising V _H -CDR2, wherein each X _n is independently any A formula EX _n X ₉ GX ₁₀ X _n X _n DX _n (natural amino acid, X ₉ is M or V, and X ₁₀ is A or G
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 part thereof is _VL- CDR1, _VL- CDR2 and _VL- C.
DR3 can be included. In such embodiments, the formula X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 3) wherein each X _n is independently any natural amino acid and X ₁₁ is H or Y.
17) represents a consensus sequence comprising V _L -CDR1, X ₁₂ is R or K, X _1
The formula GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), wherein ₃ is A or P, represents a consensus sequence comprising V _L -CDR2, and the formula STWDSX ₁₄ LSAX ₁₅ (SEQ ID NO: 319) is represented by V _L −
Represents a consensus sequence that includes 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. Less than 20 pM, less than 30 pM, less than 40 pM, 50 pM
Less than, less than 60 pM, less than 70 pM, less than 80 pM, less than 90 pM or less than 100 pM
Neutralize hGM-CSF activity to have a C50 value in the growth inhibition assay. In a specific embodiment, the claimed anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof is less than 40 pM or 3 as determined in a TF-1 proliferation assay with ED80.
It has an IC50 value that is less than 0 pM. In a specific embodiment, the claimed anti-hGM-
The 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 TF-1 proliferation assay with ED80. In a further embodiment, the present 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 is hG
When TF-1 cells are expanded by induction of M-CSF, a concentration of about 50 at a concentration of about 14 pM.
It inhibits the growth of% TF-1 cells.

As demonstrated in more detail in the examples, both EV1018 and EV1019 are hGM
-Effective in neutralizing the physiological activity of 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. Thus, the present invention is defined by the polypeptide sequences in which the epitope of hGM-CSF is set forth in SEQ ID NO: 2 and SEQ ID NO: 3, and EV1018, EV101
Screening methods recognized by 9 or variants thereof are also contemplated. 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 is Ig.
It may be any subtype of immunoglobulin, such as an IgG class such as G 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. A non-limiting example of the variable region of the heavy chain described in this disclosure is SEQ ID NO: 1.
52-159 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 λ light chain contains one or both of the following amino acid substitutions: R100G and A153G (substitution R100G, substitution A153G, or substitution R100G and substitution A153
Containing both G). Substitutions in the amino acid sequence of an antibody chain are described herein by reference to the appropriate reference amino acid sequence, positions 100 and 1 in any light chain or fragment thereof.
The designation of 53 corresponds to the amino acid position in SEQ ID NO: 35 where R is substituted with G and A is substituted with G when the amino acid position is compared with the sequence in SEQ ID NO: 34. It should be noted that the determination by the present invention is made by comparison of SEQ ID NO: 34. 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 is R (arginine) at position 100 in the EV1018 light chain
Indicates that it is replaced by G (glycine), and A153G indicates that A (alanine) at position 153 in the EV1018 light chain is replaced by G (glycine).

In a specific embodiment, the light chain is SEQ ID NO: 34-37 and 184-221 and 364.
~ 365, selected from the group consisting of polypeptides (the amino acid residues of which are represented by these). Examples of light chain variable regions described herein are SEQ ID NOS: 184-2
01 and 364-365.
The light chain is one or a combination of two or more of these amino acid substitutions, and hGM-CSF
Other amino acid substitutions that do not substantially impair the ability of the antibody or antigen-binding fragment thereof to bind to and neutralize its activity. 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 T97A, T97V, N95D, N95E, N95K, N95Q, N93Q.
One or more amino acid substitutions may be included such as / N95T, K144R, L164A, and L165A. Substitutions in the amino acid sequence of an antibody chain are described herein by reference to the appropriate reference amino acid sequence, and to determine the position, R100G and A
It should be noted that the same principles should be applied as previously described in the context of 153G substitution. 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 is such that T (threonine) at position 97 in the EV1018 heavy chain is A (
L164A is positioned at position 16 in the EV1018 heavy chain.
4 indicates that L (leucine) in 4 is replaced by A (alanine).
The heavy chain is one or a combination of two or more of these amino acid substitutions, and hGM-CSF
Other amino acid substitutions that do not substantially impair the ability of the antibody or antigen-binding fragment thereof to bind to and neutralize its activity.

Further details regarding embodiments of the present invention are outlined below:
In one embodiment, the heavy chain is Q3E, T97A, T97V, N95D, N95E, N95.
Contains one or more, or one or a combination of amino acid substitutions selected from the group consisting of K, N95Q, N93Q / N95T, K144R, L164A, and L165A. In a further embodiment, the heavy chain is Q3E, T97A, T97V, N95D, N95E,
N95K, N95Q, N93Q / N95T, K144R, L164A, and L165A
And 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 amino acid residues SYGMH (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333). It is a binding fragment.

In a further embodiment, the V _H -CDR2 is an amino acid residue LTYHHGNRKFYADSV
An anti-hGM-CSF monoclonal antibody or hGM-CS that binds to hGM-CSF, including RG (SEQ ID NO: 5) or VIWHDGSKKYYADSVKG (SEQ ID NO: 334)
An antigen-binding fragment thereof that binds to F.
In a further embodiment, the V _H -CDR3 is an amino acid residue ESMGAINDN (SEQ ID NO: 6
) Or EWVGGTCDS (SEQ ID NO: 335) anti-h binding to hGM-CSF
A GM-CSF monoclonal antibody or an antigen-binding fragment thereof that binds to hGM-CSF.

Further embodiments of the invention are as follows:
V _L -CDR1 is the amino acid residue IGNNNNIGASHAVG (SEQ ID NO: 7) or SG
Anti-hGM that binds hGM-CSF, including NSSNIGSYAVG (SEQ ID NO: 330)
A CSF monoclonal antibody or antigen-binding fragment thereof that binds hGM-CSF.
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); _L- CDR3 is amino acid residue STWDSSLSAVV (SEQ ID NO: 9) or STWD
Anti-hGM-CSF binding to hGM-CSF comprising SRLSAVL (SEQ ID NO: 332)
A monoclonal antibody or an antigen-binding fragment thereof that binds to hGM-CSF.

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,
As shown in Table 7, it was found that the TF-1 assay showed 100-fold higher inhibition than the prior art antibody, while the binding affinity was less than 50% that of the prior art antibody. This indicates that the higher the affinity of the antibody, the higher the activity in the TF-1 assay.
In contrast to the teaching of 6/122797.

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 of the present invention or an antigen-binding fragment thereof to the culture system, or the anti-hGM of the present invention.
It can be inhibited by first reacting CSF monoclonal antibody or antigen-binding fragment thereof with hGM-CSF and then adding it to the culture system. For example, the anti-hGM-CSF monoclonal antibody of the present invention or an antigen-binding fragment thereof has hGM-
The neutralizing activity against TF-1 cells induced by CSF is about 2 ng / ml (about 1
It can have a cell growth inhibition rate of 40-60% (about 50%) compared to negative subjects (31 pg / ml hIgG) at a concentration of 4 pM).
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 allergic dermatitis, autoimmune disease, chronic obstructive pulmonary disease (COPD)
Various diseases caused by hGM-CSF, including asthma, cystic fibrosis, interstitial lung disease, related arthropathy such as rhinitis, arthritis and rheumatoid arthritis, psoriasis, myeloid leukemia, and multiple sclerosis It is expected to be applicable as a therapeutic agent to be used in small amounts or doses for the treatment of

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 Inflammation, bronchiectasis, ARD
S (adult respiratory distress syndrome) and all forms of pulmonary edema, especially COPD (chronic obstructive pulmonary disease)
Obstructive pulmonary disease selected from asthma, bronchial asthma, childhood asthma, severe asthma, acute asthma attack and chronic bronchitis, emphysema originating from COPD (chronic obstructive pulmonary disease) or α1-proteinase inhibitor deficiency, From restraint lung disease induced by occupational harmful substances such as allergic alveolitis, asbestosis or silicosis, and restraint caused by lung tumors such as cancerous lymphangiopathy, bronchoalveolar carcinoma and lymphoma Pneumonia caused by selected restrictive lung disease, such as infection by viruses, bacteria, fungi, protozoa, helminths or other pathogens, such as aspiration and left heart failure, radiation pneumonia or fibrosis, eg systemic Depending on various factors such as lupus erythematosus, systemic scleroderma or sarcoidosis, and collagen diseases such as granulomatosis Caused pneumonia, such as Beck's disease, idiopathic interstitial pneumonia or idiopathic pulmonary fibrosis (IPF), mucosidosis, bronchitis caused by bacterial or viral infection, allergic bronchitis and toxic bronchitis, bronchiectasis, Pulmonary edema, for example 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 There are, but not limited to.

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 Impedes the progression of disorders associated with, causes aberrant regression of disorders associated with abnormal expression and / or activity of human GM-CSF, or human GM-CSF
A therapeutic agent (eg, human GM-CS) that is sufficient to enhance or improve the therapeutic effect (s) of another treatment for a disorder associated with aberrant expression and / or activity of
The amount of the antibody that immunospecifically binds to F).

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 directed to one (one, at least one) anti-hGM-CSF as described herein.
A therapeutic composition comprising a monoclonal antibody or antigen-binding fragment thereof. In one embodiment, the therapeutic composition comprises the following ELYK (SEQ ID NO: 2) and TMMAS in hGM-CSF.
One or more anti-hGM-CSF monoclonal antibodies or antigen-binding fragments that recognize HYKQH (SEQ ID NO: 3), 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 type anti-hG
It may be included in combination, such as an M-CSF monoclonal antibody and one or more types or types of antigen-binding fragments thereof.

A specific embodiment is one (or more) anti-hGM-binding to hGM-CSF.
A therapeutic composition comprising a CSF monoclonal antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, the antibody or antigen-binding fragment thereof comprising: (a) (i) X ₁ is Y or H; Consensus V _H -CDR1-containing sequence FTFSX ₁ X ₂ M ₂ is G or A
H (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, 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 independently any natural amino acid, X ₉ is M or V, and X ₁₀ is A or G
A consensus V _H -CDR3 containing sequence 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 _VL − where each X _n is independently any natural amino acid and X ₁₁ is H or Y CDR1 comprising SEQ _{X n GNX n X n NIGSX 11} AVG ( SEQ ID NO: 317), (ii) X ₁₂ is R or K, consensus V _L -CDR2 containing sequence GX ₁₂ SPX ₁₃ X ₁₃ is a or P
SG (SEQ ID NO: 318), and (iii) X ₁₄ is R or S, and X ₁₅ is V or L
Consensus V _L -CDR3 containing sequence STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 31)
9), or in the case of antibody fragments, the light chain or part thereof, and this anti-hGM-
The CSF monoclonal antibody or a fragment thereof is ELYK (SEQ ID NO: 2) in hGM-CSF.
) And TMMASHYKQH (SEQ ID NO: 3).

two or more anti-hGM-CSF monoclonal antibodies that bind to hGM-CSF, hGM-
Described herein is a composition comprising two or more antigen-binding fragments thereof or combinations thereof that bind to CSF and a pharmaceutically acceptable carrier, wherein one or more anti-hGM-CSF monoclonal antibodies or one Alternatively, a plurality of its antigen-binding fragments recognize the discontinuous epitopes ELYK (SEQ ID NO: 2) and TMMASHYKQH (SEQ ID NO: 3) in hGM-CSF.
Further embodiments described herein include two or more anti-hGMs that bind hGM-CSF.
A composition comprising a CSF monoclonal antibody, two or more antigen-binding fragments thereof or combinations thereof and a pharmaceutically acceptable carrier, wherein one or more antibodies or one or more antigen-binding fragments thereof, (A) (i) 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, and 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
A consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ Y
ADSVX ₈ G (SEQ ID NO: 315), and (iii) consensus V _H -CDR3 where each X _n is independently any natural amino acid, X ₉ is M or V, and X ₁₀ is A or G.
A heavy chain containing the sequence 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) each X _n is independently optional Natural amino acids of
Consensus V _L -CDR1 containing sequence X ₁₁ is H or _{Y X n GNX n X n NIGSX} 11
AVG (SEQ ID NO: 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) Consensus V _L -CDR3 where X ₁₄ is R or S and X ₁₅ is V or L
A light chain containing the sequence STWDSX ₁₄ LSAVX ₁₅ (SEQ ID NO: 319), or in the case of an antibody fragment, the light chain or a part thereof, the anti-hGM-CSF monoclonal antibody or fragment thereof being the ELYK (sequence Number 2) and TMMASHYKQH (SEQ ID NO: 3) are recognized.

Further subjects described herein include two or more anti-hGM-Cs that bind to hGM-CSF.
At least one of the SF monoclonal antibodies is SEQ ID NO: 10-33, 38-80, 152
~ 183, 34 ~ 37, 184 ~ 221, 222 ~ 245, 320 ~ 323 and 348
A composition comprising a polypeptide selected from the group consisting of ~ 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.
For example, PCT application international publication No. WO2006 / 122797, PCT application international publication No. W
See O2007 / 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. Kits can be provided in a variety of formats, in addition to the drug (therapeutic composition)
A package or / and label for a drug or device, usually derived from a prescription from a physician, can also be included. 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,
It can be 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.

As used herein, a “pharmaceutically acceptable carrier” is any of a physiologically compatible solution, dispersion medium, coating agent, antibacterial or antifungal agent, osmotic pressure regulator, and absorption retardant. 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 are:
Small amounts of auxiliary substances such as wetting agents, emulsifying agents, preservatives, and buffering agents can be included. 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, 0.001 to
It is preferred to condition the antibody for an injection solution containing the antibody in an amount of 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 flint or amber vials,
Can be prepared in ampoules or pre-filled 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 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,
Antibodies such as controlled release formulations that can control the release of the compound are also applied with the active compound for preparation. The biocompatible polymer is biodegradable. Preparations include ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters (polyor
titer esters), and biocompatible polymers such as 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-hG of the invention
M-CSF monoclonal antibody or antigen-binding fragment thereof is formulated with one or more types of other therapeutic agents useful for treating diseases associated with high amounts of GM-CSF, or at the same time other therapeutic agents In which case the other therapeutic agent is not an antibody against 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 (
The effect of the drug (s) (second, third, etc.) depends on the time in the subject and the target cell /
As long as it overlaps in the tissue and thereby results in improved (eg, synergistic) therapeutic results, it is 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) 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 an anti-hGM-CSF monoclonal antibody specific for hGM-CSF or an 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 provides a plurality of different types of anti-hGM-CSF monoclonal antibodies of the present invention (hereinafter referred to as “multiple types”), or a plurality of types of anti-hGM-C of the present invention.
Also included is a method for enhancing activity, characterized in that a pharmaceutical composition or veterinary pharmaceutical composition comprising an SF antibody 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. An anti-hGM-CSF monoclonal antibody and an antigen-binding fragment thereof,
And / or therapeutic compositions, pharmaceutical compositions and pharmaceutical compositions comprising such antibodies and / or antigen-binding fragments thereof, and / or
H in a control, such as an individual (s) who does not have a disease, condition or disorder
A disease, disorder or condition associated with its overexpression (as compared to the expression level of GM-CSF) 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 a disease 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” refers to hG
Conditions including the same result as M-CSF overexpression should be included.

Production / Preparation In one embodiment, the present invention provides an anti-hG disclosed herein for use in medicine.
M-CSF monoclonal antibody or antigen-binding fragment thereof.
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.

ELYK (SEQ ID NO: 2) and T in hGM-CSF for the manufacture or preparation of a drug
The use of an antibody that recognizes MMASHYKQH (SEQ ID NO: 3) or an antigen-binding fragment thereof 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) 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 arbitrary X ₃ is L or V, X ₄ is T or I, X ₅ is Y or W, X ₆ is R or K, and X ₇ is F or Y , X ₈ 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: 3
15), and (iii) each X _n is independently any natural amino acid and X ₉ is M or V
And a consensus V _H -CDR3 containing sequence EX _n X ₉ GX ₁₀ X _n where X ₁₀ is A or G
A heavy chain comprising 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) each X _n is independently any natural amino acid, and X ₁₁ is H Or a consensus V _L -CDR1-containing sequence X _n GNX _n X _n NIGSX ₁₁ AVG (SEQ ID NO: 317), which is Y or
(Ii) Consensus V _L -CDR2 where X ₁₂ is R or K and X ₁₃ is A or P
Containing sequence GX ₁₂ SPX ₁₃ SG (SEQ ID NO: 318), and (iii) Consensus V _L -CDR3 containing sequence STWDSX ₁₄ LS where X ₁₄ is R or S and X ₁₅ is V or L
A light chain comprising AVX ₁₅ (SEQ ID NO: 319), or in the case of an antibody fragment, comprises a light chain or a portion thereof.

In a further aspect, the invention provides an isolated anti-hGM-CS that is useful in the manufacture of a medicament.
F antibodies or antigen-binding fragments of such antibodies are provided. These drugs are hGM-CS
Useful for the treatment of diseases, conditions or disorders associated with abnormal expression (eg, overexpression) of F. 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 consists of ELYK (SEQ ID NO: 2) and TMM in hGM-CSF.
Recognizes ASHYKQH (SEQ ID NO: 3).
In certain embodiments, the antibody or antigen-binding fragment thereof comprises (a) (i) X ₁ is Y or H
And a consensus V _H -CDR1-containing sequence FTFSX ₁ X ₂ MH where X ₂ is G or A
(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, Consensus V _H -CDR2 containing sequence X ₃ X ₄ X ₅ HX _n GX _n X ₆ KX ₇ YADSVX ₈ G wherein X 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 wherein} each X _n is independently any natural amino acid, X ₉ is M or V, and X ₁₀ is A or G 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) X ₁₂ is R or K
And a consensus V _L -CDR2 containing sequence GX ₁₂ SPX ₁₃ S wherein X ₁₃ is A or P
G (SEQ ID NO: 318), and (iii) a 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 in the case of antibody fragments, part of the light chain. In one embodiment, the antibody or antigen-binding fragment thereof has a hGM-CSF up to 400 pM, in another embodiment about 16
Bind with a K _D of less than 0 pM (such EV1018 or EV1019), K _D is determined in accordance with the techniques described in Example.
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 obtained 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 B-lymphocytes were obtained from the blood of patients suffering from idiopathic alveolar proteinosis (IPAP) and having high levels of anti-hGM-CSF monoclonal antibodies in the serum. 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
Maintain 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. Preferably, an ELISA is used 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. Then, protein A-Sepharose or protein G-Sepharose (New Jersey, Piscataway, Pharmacia C
orp. The protein is purified by affinity chromatography using 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, anti-hGM
-The affinity of the CSF monoclonal antibody is 10 to 100 times higher than the antibody 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.

The table below shows EV1018 heavy and light chain variants and EV1019.
Heavy chain variants 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.
Allows the identification of all possible combinations of heavy and light chains for 8 and EV1019 respectively. 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. Anti-hGM-CS produced
F monoclonal antibodies or antigen-binding fragments are obtained by methods known in the art,
It can be recovered from the host cell or host cell culture.

More specifically, the process comprises at least V _H -CDR1, V _H -CDR2 in the host cell.
And V _H -CDR3, and V _L -CDR1, V _L -CDR2 and V _L -CDR3. The methods are V _H -CDR1, V _H -CDR2, V _H -CDR3, V _L -CDR.
1, DNA encoding V _L -CDR2 and V _L -CDR3 is introduced into an appropriate host cell, and the generated host cells (V _H -CDR1, V _H -CDR2, V _H -CDR3, V _L -CDR1,
Conditions suitable for expression of DNA (generation of coding peptide) and formation / construction of anti-hGM-CSF monoclonal antibody or antigen-binding fragment) (host cells containing DNA encoding _VL- CDR2, _VL- CDR3) Maintaining under, thereby producing an anti-hGM-CSF monoclonal antibody or antigen-binding fragment. In a specific embodiment, anti-hGM-C
The SF monoclonal 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 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 a consensus V _H -CDR2 containing sequence X ₃ X _{4 wherein} F is F or Y and X ₈ is R or K
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 a consensus V _H -CDR3-containing sequence EX _n X ₉ GX ₁₀ X _n X _n DX _n (SEQ ID NO: 316) that is or G, or a portion of the heavy chain in the case of an antibody fragment, and (b) (i ) is any naturally occurring amino acid each X _n is independently consensus V _L -CDR1 containing sequence X ₁₁ is H or _{Y X n GNX n X n NIGSX} 11 AVG ( SEQ ID NO: 317), (ii) X ₁₂ Is a consensus V _L -CDR2 containing sequence GX ₁₂ SPX ₁₃ SG (wherein R is K or 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 an antibody fragment, In the case of comprising a light chain or a part thereof, and the anti-hGM-CSF monoclonal antibody or the produced antigen-binding fragment binds to hGM-CSF. V _H -CDR
1, V _H -CDR2, V _H -CDR3, V _L -CDR1, V _L -CDR2 and V _L -CDR
DNA encoding 3 may be one or multiple units (eg, all of the V _H / V _L components may be encoded by one DNA, some or all of the components may be distinct May be encoded by 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 is SEQ ID NO: 10-33, 38-80 and 16
0 to 183; can be selected from the group consisting of 222 to 245, 320 and 322;
The complete light chain can be selected from the group consisting of SEQ ID NOs: 34-37, 202-221, 321, and 323. In one embodiment, the complete heavy chain is SEQ ID NO: 10-33, 38-80.
And 160-183; 222-245, 320 and 322,
The complete light chain is selected from the group consisting of SEQ ID NOs: 34-37, 202-221, 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 SYGM
H (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333), and V _H -CDR2 is LTY
HHGNRKFYADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVK
G (SEQ ID NO: 334) and V _H -CDR3 is ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS (SEQ ID NO: 335).

Further subject matter of the present specification, V _L -CDR1, a vector comprising DNA encoding the V _L -CDR2, and V _L -CDR3, V _L -CDR1 is IGNNNNIGSHAVG (
SEQ ID NO: 7) or SGNSSNIGYASV (SEQ ID NO: 330), V _L -CD
R2 is GRSPPS (SEQ ID NO: 8) or GKSPAS (SEQ ID NO: 331), and V _L
-CDR3 is STWDDSLSAVV (SEQ ID NO: 9) or STWDDSLSAVL (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 DN capable of hybridizing with the previously described DNA under stringent conditions
A.
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 is a light chain, EV1018-
wt-original (SEQ ID NO: 34) and the following heavy chain variants: EV1018 (SEQ ID NO: 1)
0), 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: 1)
6), EV1018-N95Q IgG1KO (SEQ ID NO: 17), EV1018-N93
Q-N95T IgG1KO (SEQ ID NO: 18), EV1018-wt IgG1-BI (
SEQ ID NO: 19), EV1018-T97A IgG1-BI (SEQ ID NO: 20), EV10
18-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: 2
7), EV1018-T97V IgG1-original-constant (SEQ ID NO: 28), EV1
018-N95D IgG1-original-constant (SEQ ID NO: 29), EV1018-N9
5E IgG1-original-constant (SEQ ID NO: 30), EV1018-N95K IgG
1-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: 3
8), EV1018-T97A-IgG1-KO-QVQL (SEQ ID NO: 39), EV10
18-T97V-IgG1-KO-QVQL (SEQ ID NO: 40), EV1018-N95D
-IgG1-KO-QVQL (SEQ ID NO: 41), EV1018-N95E IgG1-K
O-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-N95T IgG1-KO-QVQL (SEQ ID NO: 45), E
V1018-wt IgG1-QVQL-BI (SEQ ID NO: 46), EV1018-T97
A 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), EV101
8-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), E
V1018-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-I
gG4 (SEQ ID NO: 64), EV1018-T97A-IgG4 (SEQ ID NO: 65), EV1
018-T97V-IgG4 (SEQ ID NO: 66), EV1018-N95D-IgG4 (SEQ ID NO: 67), EV1018-N95E-IgG4 (SEQ ID NO: 68), EV1018-N
95K-IgG4 (SEQ ID NO: 69), EV1018-N95Q-IgG4 (SEQ ID NO: 70)
), EV1018-N93Q-N95T-IgG4 (SEQ ID NO: 71), EV1018-I
gG4-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 one of EV1018-N93Q-N95T-IgG4-SP (SEQ ID NO: 80).

In some embodiments, the antibody or antigen-binding fragment thereof is a light chain, EV1018-
wt-BI (SEQ ID NO: 35), and the following heavy chain variants: EV1018 (SEQ ID NO: 10),
EV1018-wt-IgG1KO (SEQ ID NO: 11), EV1018-T97A-IgG
1 KO (SEQ ID NO: 12), EV1018-T97V-IgG1 KO (SEQ ID NO: 13), E
V1018-N95D-IgG1KO (SEQ ID NO: 14), EV1018-N95E Ig
G1KO (SEQ ID NO: 15), EV1018-N95K IgG1KO (SEQ ID NO: 16),
EV1018-N95Q IgG1KO (SEQ ID NO: 17), EV1018-N93Q-N
95T 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), EV1
018-N95K IgG1-BI (SEQ ID NO: 24), EV1018-N95Q IgG
1-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-Ig
G1-KO-QVQL (SEQ ID NO: 41), EV1018-N95E IgG1-KO-Q
VQL (SEQ ID NO: 42), EV1018-N95K IgG1-KO-QVQL (SEQ ID NO: 43), EV1018-N95Q IgG1-KO-QVQL (SEQ ID NO: 44), EV
1018-N93Q-N95T IgG1-KO-QVQL (SEQ ID NO: 45), EV10
18-wt IgG1-QVQL-BI (SEQ ID NO: 46), EV1018-T97A I
gG1-QVQL-BI (SEQ ID NO: 47), EV1018-T97V IgG1-QVQ
L-BI (SEQ ID NO: 48), EV1018-N95D IgG1-QVQL-BI (SEQ ID NO: 49), EV1018-N95E IgG1-QVQL-BI (SEQ ID NO: 50), E
V1018-N95K IgG1-QVQL-BI (SEQ ID NO: 51), EV1018-N
95Q IgG1-QVQL-BI (SEQ ID NO: 52), EV1018-N93Q-N95
T IgG1-QVQL-BI (SEQ ID NO: 53), EV1018-IgG2 (SEQ ID NO: 5)
4), EV1018-wt-IgG2 (SEQ ID NO: 55), EV1018-T97A-Ig
G2 (SEQ ID NO: 56), EV1018-T97V-IgG2 (SEQ ID NO: 57), EV10
18-N95D-IgG2 (SEQ ID NO: 58), EV1018-N95E-IgG2 (SEQ ID NO: 59), EV1018-N95K-IgG2 (SEQ ID NO: 60), EV1018-N9
5Q-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), E
V1018-N93Q-N95T-IgG4 (SEQ ID NO: 71), EV1018-IgG4
-SP (SEQ ID NO: 72), EV1018-wt-IgG4-SP (SEQ ID NO: 73), EV
1018-T97A-IgG4-SP (SEQ ID NO: 74), EV1018-T97V-Ig
G4-SP (SEQ ID NO: 75), EV1018-N95D-IgG4-SP (SEQ ID NO: 76)
), EV1018-N95E-IgG4-SP (SEQ ID NO: 77), EV1018-N95
K-IgG4-SP (SEQ ID NO: 78), EV1018-N95Q-IgG4-SP (SEQ ID NO: 79) and EV1018-N93Q-N95T-IgG4-SP (SEQ ID NO: 80)
One of these.

In some embodiments, the antibody or antigen-binding fragment thereof is a light chain, EV1018-
wt-B12 (G1) (SEQ ID NO: 36) and the following heavy chain variants: EV1018 (SEQ ID NO: 10), EV1018-wt-IgG1 KO (SEQ ID NO: 11), EV1018-T97
A-IgG1KO (SEQ ID NO: 12), EV1018-T97V-IgG1KO (SEQ ID NO: 13), EV1018-N95D-IgG1KO (SEQ ID NO: 14), EV1018-N9
5E IgG1KO (SEQ ID NO: 15), EV1018-N95K IgG1KO (SEQ ID NO: 16), EV1018-N95Q IgG1KO (SEQ ID NO: 17), EV1018-N
93Q-N95T IgG1KO (SEQ ID NO: 18), EV1018-wt IgG1-B
I (SEQ ID NO: 19), EV1018-T97A IgG1-BI (SEQ ID NO: 20), EV
1018-T97V IgG1-BI (SEQ ID NO: 21), EV1018-N95D Ig
G1-BI (SEQ ID NO: 22), EV1018-N95E IgG1-BI (SEQ ID NO: 23)
), EV1018-N95K IgG1-BI (SEQ ID NO: 24), EV1018-N95
Q 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), E
V1018-N95D IgG1-original-constant (SEQ ID NO: 29), EV1018-
N95E IgG1-original-constant (SEQ ID NO: 30), EV1018-N95K I
gG1-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), EV
1018-T97V-IgG1-KO-QVQL (SEQ ID NO: 40), EV1018-N9
5D-IgG1-KO-QVQL (SEQ ID NO: 41), EV1018-N95E IgG1
-KO-QVQL (SEQ ID NO: 42), EV1018-N95K IgG1-KO-QVQ
L (SEQ ID NO: 43), EV1018-N95Q IgG1-KO-QVQL (SEQ ID NO: 4
4), EV1018-N93Q-N95T IgG1-KO-QVQL (SEQ ID NO: 45)
EV1018-wt IgG1-QVQL-BI (SEQ ID NO: 46), EV1018-T
97A IgG1-QVQL-BI (SEQ ID NO: 47), EV1018-T97V IgG
1-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), EV1
018-N95Q IgG1-QVQL-BI (SEQ ID NO: 52), EV1018-N93
Q-N95T IgG1-QVQL-BI (SEQ ID NO: 53), EV1018-IgG2 (
SEQ ID NO: 54), EV1018-wt-IgG2 (SEQ ID NO: 55), EV1018-T9
7A-IgG2 (SEQ ID NO: 56), EV1018-T97V-IgG2 (SEQ ID NO: 57)
EV1018-N95D-IgG2 (SEQ ID NO: 58), EV1018-N95E-Ig
G2 (SEQ ID NO: 59), EV1018-N95K-IgG2 (SEQ ID NO: 60), EV10
18-N95Q-IgG2 (SEQ ID NO: 61), EV1018-N93Q-N95T-Ig
G2 (SEQ ID NO: 62), EV1018-IgG4 (SEQ ID NO: 63), EV1018-wt
-IgG4 (SEQ ID NO: 64), EV1018-T97A-IgG4 (SEQ ID NO: 65), E
V1018-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: 7)
3), EV1018-T97A-IgG4-SP (SEQ ID NO: 74), EV1018-T9
7V-IgG4-SP (SEQ ID NO: 75), EV1018-N95D-IgG4-SP (SEQ ID NO: 76), EV1018-N95E-IgG4-SP (SEQ ID NO: 77), EV101
8-N95K-IgG4-SP (SEQ ID NO: 78), EV1018-N95Q-IgG4-
One of 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 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-IgG1 KO (SEQ ID NO: 11), EV1018-T9
7A-IgG1KO (SEQ ID NO: 12), EV1018-T97V-IgG1KO (SEQ ID NO: 13), EV1018-N95D-IgG1KO (SEQ ID NO: 14), EV1018-N
95E 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), E
V1018-T97V IgG1-BI (SEQ ID NO: 21), EV1018-N95D I
gG1-BI (SEQ ID NO: 22), EV1018-N95E IgG1-BI (SEQ ID NO: 2)
3), EV1018-N95K IgG1-BI (SEQ ID NO: 24), EV1018-N9
5Q 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), E
V1018-T97V-IgG1-KO-QVQL (SEQ ID NO: 40), EV1018-N
95D-IgG1-KO-QVQL (SEQ ID NO: 41), EV1018-N95E IgG
1-KO-QVQL (SEQ ID NO: 42), EV1018-N95K IgG1-KO-QV
QL (SEQ ID NO: 43), EV1018-N95Q IgG1-KO-QVQL (SEQ ID NO: 44), EV1018-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 Ig
G1-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), EV
1018-N95Q IgG1-QVQL-BI (SEQ ID NO: 52), EV1018-N9
3Q-N95T IgG1-QVQL-BI (SEQ ID NO: 53), EV1018-IgG2
(SEQ ID NO: 54), EV1018-wt-IgG2 (SEQ ID NO: 55), EV1018-T
97A-IgG2 (SEQ ID NO: 56), EV1018-T97V-IgG2 (SEQ ID NO: 57)
), EV1018-N95D-IgG2 (SEQ ID NO: 58), EV1018-N95E-I
gG2 (SEQ ID NO: 59), EV1018-N95K-IgG2 (SEQ ID NO: 60), EV1
018-N95Q-IgG2 (SEQ ID NO: 61), EV1018-N93Q-N95T-I
gG2 (SEQ ID NO: 62), EV1018-IgG4 (SEQ ID NO: 63), EV1018-w
t-IgG4 (SEQ ID NO: 64), EV1018-T97A-IgG4 (SEQ ID NO: 65),
EV1018-T97V-IgG4 (SEQ ID NO: 66), EV1018-N95D-IgG
4 (SEQ ID NO: 67), EV1018-N95E-IgG4 (SEQ ID NO: 68), EV101
8-N95K-IgG4 (SEQ ID NO: 69), EV1018-N95Q-IgG4 (SEQ ID NO: 70), EV1018-N93Q-N95T-IgG4 (SEQ ID NO: 71), EV101
8-IgG4-SP (SEQ ID NO: 72), EV1018-wt-IgG4-SP (SEQ ID NO: 73), EV1018-T97A-IgG4-SP (SEQ ID NO: 74), EV1018-T
97V-IgG4-SP (SEQ ID NO: 75), EV1018-N95D-IgG4-SP (
SEQ ID NO: 76), EV1018-N95E-IgG4-SP (SEQ ID NO: 77), EV10
18-N95K-IgG4-SP (SEQ ID NO: 78), EV1018-N95Q-IgG4
-One of SP (SEQ ID NO: 79) and EV1018-N93Q-N95T-IgG4-SP (SEQ ID NO: 80)

The present invention provides the antibody EV1019 itself and a number of variants of the EV1019 antibody as summarized 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 is wild type EV1019.
Contains one or more amino acid substitutions to the light chain sequence. In some embodiments,
Each heavy and light chain contains one or more amino acid substitutions.

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019 (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 (SEQ ID NO: 208), EV1019-N25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N25T-B
I2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV1019-
N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), E
V1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV1019-S27
G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219), EV10
19-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (SEQ ID NO: 2)
21) comprising a light chain selected from the group consisting of:

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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: 2)
05), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2
(SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N2
5G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1
019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 21)
2), 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
-Comprising a light chain selected from the group consisting of BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105G (SEQ ID NO: 163), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-B
I2 (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-B
I2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-
N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), E
V1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27
N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV10
19-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219)
), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2
A light chain selected from the group consisting of (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105G-IgG1-BI (SEQ ID NO: 164), and EV1019-wt-
Original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1
019-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), EV101
9-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-N
25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV
1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 2)
17), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2
(SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-
A light chain selected from the group consisting of S27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105G-IgG1KO (SEQ ID NO: 165), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105S (SEQ ID NO: 166), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-B
I2 (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-B
I2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-
N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), E
V1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27
N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV10
19-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219)
), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2
A light chain selected from the group consisting of (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105S-IgG1-BI (SEQ ID NO: 167), and EV1019-wt-
Original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1
019-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), EV101
9-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-N
25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV
1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 2)
17), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2
(SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-
A light chain selected from the group consisting of S27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105S-IgG1KO (SEQ ID NO: 168), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105A (SEQ ID NO: 169), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-B
I2 (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-B
I2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-
N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), E
V1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27
N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV10
19-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219)
), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2
A light chain selected from the group consisting of (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105A-IgG1-BI (SEQ ID NO: 170), and EV1019-wt-
Original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1
019-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), EV101
9-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-N
25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV
1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 2)
17), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2
(SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-
A light chain selected from the group consisting of S27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105A-IgG1KO (SEQ ID NO: 171), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105Q (SEQ ID NO: 172), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-B
I2 (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-B
I2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-
N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), E
V1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27
N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV10
19-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219)
), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2
A light chain selected from the group consisting of (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105Q-IgG1-BI (SEQ ID NO: 173), and EV1019-wt-
Original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1
019-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), EV101
9-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-N
25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV
1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 2)
17), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2
(SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-
A light chain selected from the group consisting of S27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105Q-IgG1KO (SEQ ID NO: 174), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105T (SEQ ID NO: 175), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-B
I2 (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-B
I2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-
N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), E
V1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27
N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV10
19-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219)
), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2
A light chain selected from the group consisting of (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105T-IgG1-BI (SEQ ID NO: 176), and EV1019-wt-
Original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1
019-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), EV101
9-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-N
25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV
1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 2)
17), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2
(SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-
A light chain selected from the group consisting of S27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105T-IgG1KO (SEQ ID NO: 177), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105M (SEQ ID NO: 178), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-B
I2 (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-B
I2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-
N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), E
V1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27
N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV10
19-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219)
), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2
A light chain selected from the group consisting of (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105M-IgG1-BI (SEQ ID NO: 179), and EV1019-wt-
Original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1
019-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), EV101
9-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-N
25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV
1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 2)
17), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2
(SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-
A light chain selected from the group consisting of S27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105M-IgG1KO (SEQ ID NO: 180), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105L (SEQ ID NO: 181), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-B
I2 (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-B
I2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-
N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), E
V1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27
N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV10
19-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219)
), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2
A light chain selected from the group consisting of (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105L-IgG1-BI (SEQ ID NO: 182), and EV1019-wt-
Original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1
019-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), EV101
9-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-N
25Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV
1019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 2)
17), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2
(SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-
A light chain selected from the group consisting of S27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105L-IgG1KO (SEQ ID NO: 183), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-IgG2 (SEQ ID NO: 222), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV1019-wt-BI
2 (SEQ ID NO: 204), EV1019-wt-original-constant (SEQ ID NO: 205), E
V1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N25G-BI
2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1019-N
25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 212), EV
1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q (SEQ ID NO: 2)
14), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1019-S27N
(SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV101
9-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 219)
, EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-BI2 (
A light chain selected from the group consisting of SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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), EV101
9-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-S
27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 217), EV
1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (SEQ ID NO: 2)
19), EV1019-S27A (SEQ ID NO: 220) and EV1019-S27A-B
A light chain selected from the group consisting of I2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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: 2)
05), EV1019-N25S (SEQ ID NO: 206), EV1019-N25S-BI2
(SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N2
5G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV1
019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 21)
2), 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
-Comprising a light chain selected from the group consisting of BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105G-IgG4SP (SEQ ID NO: 227), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105S-IgG4SP (SEQ ID NO: 230), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105A-IgG4SP (SEQ ID NO: 233), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105Q-IgG4SP (SEQ ID NO: 236), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105T-IgG4SP (SEQ ID NO: 239), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105M-IgG4SP (SEQ ID NO: 242), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-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-BI
2 (SEQ ID NO: 207), EV1019-N25G (SEQ ID NO: 208), EV1019-N
25G-BI2 (SEQ ID NO: 209), EV1019-N25T (SEQ ID NO: 210), EV
1019-N25T-BI2 (SEQ ID NO: 211), EV1019-N25R (SEQ ID NO: 2)
12), EV1019-N25R-BI2 (SEQ ID NO: 213), EV1019-N25Q
(SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV101
9-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-S27.
A light chain selected from the group consisting of A-BI2 (SEQ ID NO: 221).

In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a heavy chain, EV1.
019-C105L-IgG4SP (SEQ ID NO: 245), and EV1019-wt-original (SEQ ID NO: 202), EV1019-wt-BI (SEQ ID NO: 203), EV10
19-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-N2
5Q (SEQ ID NO: 214), EV1019-N25Q-BI2 (SEQ ID NO: 215), EV1
019-S27N (SEQ ID NO: 216), EV1019-S27N-BI2 (SEQ ID NO: 21)
7), EV1019-S27G (SEQ ID NO: 218), EV1019-S27G-BI2 (
SEQ ID NO: 219), EV1019-S27A (SEQ ID NO: 220) and EV1019-S
A light chain selected from the group consisting of 27A-BI2 (SEQ ID NO: 221).

In addition, the variable region of the heavy chain and the variable region of the light chain combine to bind to the antigen.
Fragments may be produced. 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), EV
1018-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 (
Any one of SEQ ID NO: 361), EV1018-VH-N95Q IgG1-original-constant (SEQ ID NO: 362), and EV1018-VH-N93Q-N95T IgG1-original-constant (SEQ ID NO: 363) are used as light chain variable regions EV1018-VL-wt-
In combination with the original (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), EV
1018-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 (
Any one of SEQ ID NO: 361), EV1018-VH-N95Q IgG1-original-constant (SEQ ID NO: 362), and EV1018-VH-N93Q-N95T IgG1-original-constant (SEQ ID NO: 363), 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-N2
5G (SEQ ID NO: 189), EV1019-VL-N25T (SEQ ID NO: 190), EV10
19-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: 194), EV1019-VL-BI-N25Q (SEQ ID NO: 195), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-B
I-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198),
EV1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27
A light chain variable region selected from the group consisting of A (SEQ ID NO: 200), EV1019-VL-BI-S27A (SEQ ID NO: 201).

In some embodiments, the antigen binding fragment of EV1019 is a heavy chain variable region, EV1.
019-VH-C105G (SEQ ID NO: 153), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 185), EV1019-VL-N
25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187),
EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25
G (SEQ ID NO: 189), EV1019-VL-N25T (SEQ ID NO: 190), EV101
9-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: 194), EV1019-VL-BI-N25Q (SEQ ID NO: 1
95), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI
-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), E
V1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A
(SEQ ID NO: 200), comprising a light chain variable region selected from the group consisting of EV1019-VL-BI-S27A (SEQ ID NO: 201).

In some embodiments, the antigen binding fragment of EV1019 is a heavy chain variable region, EV1.
019-VH-C105S (SEQ ID NO: 154), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 185), EV1019-VL-N
25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187),
EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25
G (SEQ ID NO: 189), EV1019-VL-N25T (SEQ ID NO: 190), EV101
9-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: 194), EV1019-VL-BI-N25Q (SEQ ID NO: 1
95), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI
-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), E
V1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A
(SEQ ID NO: 200), comprising a light chain variable region selected from the group consisting of EV1019-VL-BI-S27A (SEQ ID NO: 201).

In some embodiments, the antigen binding fragment of EV1019 is a heavy chain variable region, EV1.
019-VH-C105A (SEQ ID NO: 155), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 185), EV1019-VL-N
25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187),
EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25
G (SEQ ID NO: 189), EV1019-VL-N25T (SEQ ID NO: 190), EV101
9-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: 194), EV1019-VL-BI-N25Q (SEQ ID NO: 1
95), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI
-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), E
V1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A
(SEQ ID NO: 200), comprising a light chain variable region selected from the group consisting of EV1019-VL-BI-S27A (SEQ ID NO: 201).

In some embodiments, the antigen binding fragment of EV1019 is a heavy chain variable region, EV1.
019-VH-C105T (SEQ ID NO: 156), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 185), EV1019-VL-N
25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187),
EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25
G (SEQ ID NO: 189), EV1019-VL-N25T (SEQ ID NO: 190), EV101
9-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: 194), EV1019-VL-BI-N25Q (SEQ ID NO: 1
95), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI
-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), E
V1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A
(SEQ ID NO: 200), comprising a light chain variable region selected from the group consisting of EV1019-VL-BI-S27A (SEQ ID NO: 201).

In some embodiments, the antigen binding fragment of EV1019 is a heavy chain variable region, EV1.
019-VH-C105M (SEQ ID NO: 157), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 185), EV1019-VL-N
25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187),
EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25
G (SEQ ID NO: 189), EV1019-VL-N25T (SEQ ID NO: 190), EV101
9-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: 194), EV1019-VL-BI-N25Q (SEQ ID NO: 1
95), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI
-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), E
V1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A
(SEQ ID NO: 200), comprising a light chain variable region selected from the group consisting of EV1019-VL-BI-S27A (SEQ ID NO: 201).

In some embodiments, the antigen binding fragment of EV1019 is a heavy chain variable region, EV1.
019-VH-C105Q (SEQ ID NO: 158), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 185), EV1019-VL-N
25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187),
EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25
G (SEQ ID NO: 189), EV1019-VL-N25T (SEQ ID NO: 190), EV101
9-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: 194), EV1019-VL-BI-N25Q (SEQ ID NO: 1
95), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI
-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), E
V1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A
(SEQ ID NO: 200), comprising a light chain variable region selected from the group consisting of EV1019-VL-BI-S27A (SEQ ID NO: 201).

In some embodiments, the antigen binding fragment of EV1019 is a heavy chain variable region, EV1.
019-VH-C105L (SEQ ID NO: 159), and EV1019-VL-wt (SEQ ID NO: 184), EV1019-VL-BI (SEQ ID NO: 185), EV1019-VL-N
25S (SEQ ID NO: 186), EV1019-VL-BI-N25S (SEQ ID NO: 187),
EV1019-VL-N25G (SEQ ID NO: 188), EV1019-VL-BI-N25
G (SEQ ID NO: 189), EV1019-VL-N25T (SEQ ID NO: 190), EV101
9-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: 194), EV1019-VL-BI-N25Q (SEQ ID NO: 1
95), EV1019-VL-S27N (SEQ ID NO: 196), EV1019-VL-BI
-S27N (SEQ ID NO: 197), EV1019-VL-S27G (SEQ ID NO: 198), E
V1019-VL-BI-S27G (SEQ ID NO: 199), EV1019-VL-S27A
(SEQ ID NO: 200), comprising a light chain variable region selected from the group consisting of EV1019-VL-BI-S27A (SEQ ID NO: 201).

Variations within the present invention The present invention relates to full-length anti-hGM-CSF antibodies or antigen-binding portions thereof, or anti-hGM-
When two or more types of CSF antibody portions are administered simultaneously, they are included. 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 SEQ ID NOs: 348-363, SEQ ID NOs: 184-201, and SEQ ID NO: 1
Obtained by any well-known technique based on the amino acid sequence of 52-159).
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 claimed.
It is constructed in a manner known from the scope creation guidelines. Because granted by this patent application
Do you think it would be useful to illustrate in the specification the teachings that can be derived from the scope of protection
That's it.
Item 1. One embodiment provides for ELYK (SEQ ID NO: 2) in hGM-CSF (SEQ ID NO: 1).
And TMMASHYKQH (SEQ ID NO: 3), an isolated anti-hGM-CSF mono
A clonal antibody or antigen-binding fragment thereof.
Item 2. Another embodiment is:
  (A) Consensus V_H-CDR1-containing sequences, consensus V_H-CDR2 containing sequences and
Consensus V_HA heavy chain comprising a CDR3-containing sequence,
    (I) Consensus V_H-CDR1 containing sequence is FTFSX₁X₂MH (SEQ ID NO: 3
14), where X₁Is Y or H and X₂Is G or A,
    (Ii) Consensus V_HThe CDR2-containing sequence is X_ThreeX_FourX_FiveHX_nGX_nX₆KX₇Y
ADSVX₈G (SEQ ID NO: 315), wherein each X_nIs independently any natural amino acid
Yes, X_ThreeIs L or V and X_FourIs T or I and X_FiveIs Y or W and X₆But
R or K, X₇Is F or Y and X₈Is R or K;
    (Iii) Consensus V_H-CDR3 containing sequence is EX_nX₉GX_TenX_nX_nDX_n(
SEQ ID NO: 316), wherein each X_nAre independently any natural amino acid and X₉Is M
Or V and X_TenA heavy chain wherein A or G; and
  (B) Consensus V_L-CDR1-containing sequences, consensus V_L-CDR2 containing sequences and
Consensus V_LA light chain comprising a CDR3-containing sequence,
    (I) Consensus V_LThe CDR1-containing sequence is X_nGNX_nX_nNIGSX₁₁AVG
(SEQ ID NO: 317), wherein each X_nAre independently any natural amino acid and X₁₁But
H or Y,
    (Ii) Consensus V_L-CDR2 containing sequence is GX₁₂SPX₁₃SG (SEQ ID NO:
318), where X₁₂Is R or K and X₁₃Is A or P,
    (Iii) Consensus V_L-CDR3 containing sequence is STWDSX₁₄LSAVX₁₅
(SEQ ID NO: 319), wherein X₁₄Is R or S and X₁₅Light is V or L
chain
An isolated anti-hGM-CSF monoclonal antibody or an anti-antibody thereof,
Original binding fragment. In other embodiments, the antibody or antigen-binding fragment of item 1 or item 2 is
HGM-CSF preferably with a K of less than 450 pM_DBind specifically.
  Item 3. In another embodiment, in addition to the properties listed in item 1 or 2, human GM-CSF
Less than 400 pM K_DOr the antigen binding thereof disclosed in item 1 or 2
It is a fragment.
Item 4. Another embodiment is K_DOr an antibody disclosed in item 3, wherein is less than 160 pM
An antigen-binding fragment thereof.
Item 5. Another embodiment neutralizes hGM-CSF activity and thus TF-1 proliferation
IC50 value of the antibody or antigen-binding fragment thereof is 10
The antibody or antigen-binding fragment thereof disclosed in any one of items 1 to 4, which is less than 0 pM
is there.
Item 6. Another embodiment is:
  IC50 value is less than 40 pM or less than 30 pM or less than 25 pM.
The indicated antibody or antigen-binding fragment thereof. Further preferred antibodies of the present invention or antigens thereof
The IC50 value of the binding fragment is 20 p as measured by ED80 in the TF-1 proliferation assay.
Less than M, less than 25 pM, less than 30 pM or less than 40 pM.
Item 7. Another embodiment is the antibody disclosed in item 5, wherein the IC50 value is less than 20 pM.
Or an antigen-binding fragment thereof.
Item 8. In another embodiment, the heavy chain is gamma 1 (γ₁), Gamma 2 (γ₂), Gamma 3 (γ
_Three) And gamma 4 (γ_Four) Disclosed in any of items 1 to 7 selected from the group consisting of
An antibody or antigen-binding fragment thereof.
Item 9. In another embodiment, the heavy chain is SEQ ID NO: 10-33, 38-80, 160-183.
, 222-244 and 245, having the amino acid sequence selected from the group consisting of
The antibody or antigen-binding fragment thereof disclosed in any of -8.
Item 10. Another embodiment is disclosed in any of items 1-9, wherein the light chain is a lambda light chain.
Antibody or antigen-binding fragment thereof.
Item 11. In another embodiment, the lambda light chain has the following amino acid substitution: R100G or A
The antibody or antigen binding thereof disclosed in item 10 comprising at least one or both of 153G
It is a combined fragment.
Item 12. In another embodiment, the light chain is SEQ ID NO: 34-37, 202-220 and 22
Disclosed in any of items 1-11 having an amino acid sequence selected from the group consisting of 1
An antibody or antigen-binding fragment thereof.
Item 13. Another embodiment is disclosed in any of items 1-9, wherein the light chain is a kappa light chain.
Antibody or antigen-binding fragment thereof.
Item 14. In another embodiment, the heavy chain is gamma 1 (γ₁), Gamma 2 (γ₂), Gamma 3 (
γ_Three) And gamma 4 (γ_Four), Wherein the light chain is a lambda light chain
The antibody or antigen-binding fragment thereof disclosed in any one of Items 1-13.
Item 15. In another embodiment, the heavy chain is Q3E, T97A, T97V, N95D, N95
E, N95K, N95Q, N93Q / N95T, K144R, L164A and L165
Any of items 1-13, comprising one or more amino acid substitutions selected from the group consisting of A
An antibody or antigen-binding fragment thereof disclosed herein.
Item 16. Another embodiment is V_H-CDR1 is the amino acid sequence SYGMH (SEQ ID NO: 4
) Or SHAMH (SEQ ID NO: 333), the antibody disclosed in any of items 1-15
Or an antigen-binding fragment thereof.
Item 17. Another embodiment is V_H-CDR2 is the amino acid sequence LTYHHGNRKFY
ADSVRG (SEQ ID NO: 5) or VIWHGDSKKYYADSVKG (SEQ ID NO: 33)
The antibody or antigen-binding fragment thereof disclosed in any one of items 1 to 16, including 4).
Item 18. Another embodiment is V_H-CDR3 is the amino acid sequence ESMGAINDN (arranged
Any of items 1-17, including column number 6) or EWVGGTCDS (SEQ ID NO: 335)
Or an antigen-binding fragment thereof.
Item 19. Another embodiment is V_L-CDR1 is the amino acid sequence IGNNNNIGSHA
Item comprising VG (SEQ ID NO: 7) or SGNNSSNIGYASAVG (SEQ ID NO: 330)
The antibody or antigen-binding fragment thereof disclosed in any one of 1 to 18.
Item 20. Another embodiment is V_L-CDR2 is the amino acid sequence GRSPPS (SEQ ID NO:
8) or disclosed in any of items 1-19, including GKSPAS (SEQ ID NO: 331)
An antibody or antigen-binding fragment thereof.
Item 21. Another embodiment is V_L-CDR3 is the amino acid residue STWDDSLSAVV
Items 1-20, including (SEQ ID NO: 9) or STWDRLSAVL (SEQ ID NO: 332)
Or an antigen-binding fragment thereof disclosed in any of the above.
Item 22. Another embodiment comprises 6 different CDRs, wherein the sequences of the 6 CDRs are arranged.
An isolated anti-hGM-CSF monoclonal antibody or antigen binding thereof, which is a column number 4 to 9
It is a combined fragment. In other embodiments, the antibody or antigen-binding fragment of item 22 is hGM-CS.
F preferably has a K of less than 450 pM, preferably 400 pM or 160 pM._DPeculiar
Join.
Item 22A. Another embodiment is an isolated anti-hGM disclosed in any of items 2-22
-Disclosed in item 1 in combination with a CSF monoclonal antibody or antigen-binding fragment thereof
An isolated anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof.
Item 23. Another embodiment is: (i) Gamma 1 (γ₁), Gamma 2 (γ₂), Gamma 3 (γ
_Three) And gamma 4 (γ_FourOr a fragment thereof selected from the group consisting of:
i) The antibody or antigen-binding fragment thereof disclosed in item 22 comprising a light chain that is a kappa light chain.
The
Item 24. Another embodiment is: (i) Gamma 1 (γ₁), Gamma 2 (γ₂), Gamma 3 (γ
_Three) And gamma 4 (γ_FourOr a fragment thereof selected from the group consisting of:
i) The antibody or antigen-binding fragment thereof disclosed in item 22 comprising a light chain that is a lambda light chain.
The
Item 25. Another embodiment comprises 6 different CDRs, wherein the sequences of the 6 CDRs are arranged.
Isolated anti-hGM-, which specifically binds to hGM-CSF, column numbers 330-335
CSF monoclonal antibody or antigen-binding fragment thereof.
Item 26. Another embodiment is: (i) Gamma 1 (γ₁), Gamma 2 (γ₂), Gamma 3 (γ
_Three) And gamma 4 (γ_FourOr a fragment thereof selected from the group consisting of:
i) The antibody or antigen-binding fragment thereof disclosed in item 25, comprising a light chain that is a kappa light chain.
The
Item 27. Another embodiment is: (i) Gamma 1 (γ₁), Gamma 2 (γ₂), Gamma 3 (γ
_Three) And gamma 4 (γ_FourA heavy chain selected from the group consisting of :) and (ii) a lambda light chain
The antibody or antigen-binding fragment thereof disclosed in item 25, comprising a light chain.
Item 28. Another embodiment opens with any of items 1-27, further comprising a signal sequence.
The indicated antibody or antigen-binding fragment thereof.
Item 29. In another embodiment, the signal sequence is SEQ ID NO: 324, 325, and 326
29. The antibody or antigen-binding fragment thereof disclosed in item 28, selected from the group consisting of:
Item 30. Another embodiment includes a heavy chain having a heavy chain sequence and a light chain having a light chain sequence
The heavy chain sequence consists of SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-79 and 80.
The variant selected from the group wherein the light chain sequence is SEQ ID NO: 34.
hGM-CSF monoclonal antibody or antigen-binding fragment thereof.
Item 31. Another embodiment includes a heavy chain having a heavy chain sequence and a light chain having a light chain sequence
The heavy chain sequence consists of SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-79 and 80.
The variant selected from the group wherein the light chain sequence is SEQ ID NO: 35.
hGM-CSF monoclonal antibody or antigen-binding fragment thereof.
Item 32. Another embodiment includes a heavy chain having a heavy chain sequence and a light chain having a light chain sequence
The heavy chain sequence consists of SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-79 and 80.
The variant selected from the group wherein the light chain sequence is SEQ ID NO: 36.
hGM-CSF monoclonal antibody or antigen-binding fragment thereof.
Item 33. Another embodiment includes a heavy chain having a heavy chain sequence and a light chain having a light chain sequence
The heavy chain sequence consists of SEQ ID NO: 10 or SEQ ID NOs: 11-33, 38-79 and 80.
The variant selected from the group wherein the light chain sequence is SEQ ID NO: 37.
hGM-CSF monoclonal antibody or antigen-binding fragment thereof.
Item 34. Another embodiment includes a heavy chain having a heavy chain sequence and a light chain having a light chain sequence
, Wherein the heavy chain sequence is SEQ ID NO: 160 or SEQ ID NOs: 161-244 and 245
A variant thereof selected from: wherein the light chain sequence is SEQ ID NO: 202 or SEQ ID NO: 203-
An isolated anti-hGM-C, a variant thereof selected from the group consisting of 220 and 221
SF monoclonal antibody or antigen-binding fragment thereof.
Item 35. Another embodiment is the heavy chain variable region amino acid sequence SEQ ID NO: 152 or
Said heavy chain variable which is a variant thereof selected from the group consisting of numbers 153 to 158 and 159
Region, as well as the light chain variable region amino acid sequence is SEQ ID NO: 184 or SEQ ID NO: 185-2
A light chain variable region that is a variant thereof selected from the group consisting of 00 and 201,
The released anti-hGM-CSF monoclonal antibody or antigen-binding fragment thereof.
Item 36. Another embodiment is the amino acid sequence of the heavy chain variable region is SEQ ID NO: 152
The antibody or antigen-binding fragment thereof disclosed in Item 35.
Item 37. Another embodiment is the amino acid sequence of the light chain variable region is SEQ ID NO: 184.
The antibody or antigen-binding fragment thereof disclosed in Item 35.
Item 38. In another embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 152,
The antibody or antigen binding thereof disclosed in item 35, wherein the amino acid sequence of the chain variable region is 184
It is a fragment.
Item 39. In another embodiment, the antibody is an IgG₁Terms belonging to (λ) class (subclass)
The antibody or antigen-binding fragment thereof disclosed in any one of Items 35-38.
Item 40. Another embodiment is the heavy chain variable region amino acid sequence SEQ ID NO: 348 or the sequence
Said heavy chain variable which is a variant thereof selected from the group consisting of numbers 349-362 and 363
The amino acid sequence of the region and the light chain variable region is SEQ ID NO: 364 or SEQ ID NO: 365
An isolated anti-hGM-CSF monoclonal antibody comprising the light chain variable region
An antigen-binding fragment.
Item 41. Another embodiment is the amino acid sequence of the heavy chain variable region is SEQ ID NO: 348.
The antibody or antigen-binding fragment thereof disclosed in Item 40.
Item 42. Another embodiment is the amino acid sequence of the light chain variable region is SEQ ID NO: 364.
The antibody or antigen-binding fragment thereof disclosed in Item 40.
Item 43. Another embodiment is the amino acid sequence of the light chain variable region is SEQ ID NO: 365
The antibody or antigen-binding fragment thereof disclosed in Item 40.
Item 44. In another embodiment, the antibody is an IgG₁Terms belonging to (λ) class (subclass)
The antibody or antigen-binding fragment thereof disclosed in any one of Items 40-43.
Item 45. Another embodiment is the anti-hGM-CSF monoclonal disclosed in items 1-44
An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof.
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 disclosed in item 47, wherein the vector is an expression vector.
A host cell containing a vector.
Item 49. Another embodiment is (a) an antibody disclosed in any of items 1 to 44 or a
An antigen-binding fragment, and (b) one or more containers containing the antibody or antigen-binding fragment thereof
It is a kit including a container.
Item 50. Another embodiment is disclosed in any of items 1-44 for medical use.
Antibody or antigen-binding fragment thereof.
Item 51. Another embodiment is an antibody or antigen binding thereof disclosed in any of items 1-44.
A composition comprising a combined fragment and a pharmaceutically acceptable carrier.
Item 52. Another embodiment provides a plurality of said compositions, each of which binds hGM-CSF.
An antibody, a plurality of said antigen-binding fragments, or at least one of said antibodies and at least one
A second isolated antibody that binds to hGM-CSF so as to contain the antigen-binding fragment of
Is the composition disclosed in item 51, further comprising an antigen-binding fragment thereof.
Item 53. In another embodiment, at least one of the antibodies or antigen-binding fragments thereof is
From column numbers 10-80, 152-245, 320-323, 348-364 and 365
53. The composition of item 52, which is a polypeptide having a sequence selected from the group consisting of:
Item 54. Another embodiment provides a composition disclosed in any of items 51-53 and the set.
A kit comprising one or more containers containing the composition.
Item 55. Another embodiment is:
  56. The kit disclosed in item 49 or item 54, further comprising instructions.
Item 56. Another embodiment is for a disease associated with overexpression of hGM-CSF in a subject.
Or items 1-44 and 51 for making or preparing a medicament for treating a disorder
An antibody or antigen-binding fragment or composition thereof disclosed in any of -53, wherein hGM
-An antibody or antigen binding thereof that binds to CSF and can neutralize hGM-CSF activity
Fragment or composition.
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 white
Use as disclosed in item 56, selected from the group consisting of hematological disorders as well as multiple sclerosis.
Item 58. Another embodiment is for antibodies or antigen-binding fragments at doses not exceeding 500 mg.
Use as disclosed in item 56 or 57, administered to an elephant.
Item 59. Another embodiment is for a disease associated with overexpression of hGM-CSF in a subject.
Or an antibody disclosed in any of items 1-44 and 51-53 for treating a disorder.
Or an antigen-binding fragment or composition thereof, wherein the antibody or antigen-binding fragment thereof is
It is a use that 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 white
60. The use disclosed in item 59, selected from the group consisting of blood disease as well as multiple sclerosis.
Item 61. Another embodiment is for antibodies or antigen-binding fragments at doses not exceeding 500 mg.
Use as disclosed in item 59 or 60, administered to an elephant.
Item 62. Another embodiment is the hG in the polypeptides disclosed in SEQ ID NOs: 2 and 3.
An epitope of M-CSF, wherein said epitope is disclosed in item 22 or 25
Or an antigen-binding fragment thereof, and the polypeptide sequence is hGM-CSF (arranged
It is an epitope representing the discontinuous part of column number 1).
Item 63. Another embodiment is an epitope that is a discontinuous portion of human GM-CSF.
Amino acid residues 77-80 of human GM-CSF (SEQ ID NO: 1) and human GM-CSF
Comprising amino acid residues 95-104 of (SEQ ID NO: 1), SEQ ID NO: 4-9 or SEQ ID NO: 33
Anti-hGM-CSF monoclonal antibody containing 6 different CDRs disclosed in 0-335
Or an epitope recognized by its antigen-binding fragment.
Item 64. Another embodiment is an anti-hGM-CSF monochromator that binds hGM-CSF.
A method for producing an antibody or an antigen-binding fragment thereof in a host cell, wherein the antibody or its
Antigen-binding fragment is at least consensus V_H-CDR1-containing sequences, consensus V_H-CD
R2-containing sequence, consensus V_H-CDR3-containing sequences, consensus V_L-CDR1 containing distribution
Row, consensus V_LCDR2-containing sequences and consensus V_L-Includes CDR3-containing sequences
The method is
  (I) at least the consensus V_H-CDR1-containing sequences, the consensus V_H-C
DR2-containing sequence, the consensus V_H-CDR3-containing sequences, said consensus V_L-CD
R1-containing sequence, the consensus V_L-CDR2 containing sequences and said consensus V_L-C
Obtaining a host cell comprising at least one DNA sequence encoding a DR3-containing sequence.
What
    (A) Consensus V_H-CDR1 containing sequence is FTFSX₁X₂MH (SEQ ID NO: 3
14), where X₁Is Y or H and X₂Is G or A,
    (B) Consensus V_HThe CDR2-containing sequence is X_ThreeX_FourX_FiveHX_nGX_nX₆KX₇YA
DSVX₈G (SEQ ID NO: 315), wherein each X_nIs independently any natural amino acid
X_ThreeIs L or V and X_FourIs T or I and X_FiveIs Y or W and X₆Is R
Or K and X₇Is F or Y and X₈Is R or K;
    (C) Consensus V_H-CDR3 containing sequence is EX_nX₉GX_TenX_nX_nDX_n(Array
Number 316), where each X_nAre independently any natural amino acid and X₉Is M or
V and X_TenIs A or G,
    (D) Consensus V_LThe CDR1-containing sequence is X_nGNX_nX_nNIGSX₁₁AVG
(SEQ ID NO: 317), wherein each X_nAre independently any natural amino acid and X₁₁But
H or Y,
    (E) Consensus V_L-CDR2 containing sequence is GX₁₂SPX₁₃SG (SEQ ID NO: 3
18), where X₁₂Is R or K and X₁₃Is A or P,
    (F) Consensus V_L-CDR3 containing sequence is STWDSX₁₄LSAVX₁₅(Distribution
Column number 319), where X₁₄Is R or S and X₁₅Is V or L;
and
  (Ii) conditions suitable for host cells to express DNA and produce antibodies or antigen-binding fragments thereof
Incubate under conditions
It is a method including.
Item 65. In another embodiment, the at least one DNA is a heavy chain or portion thereof and
Encodes a light chain or part thereof, wherein the heavy chain or part thereof is SEQ ID NO: 10-33, 38-
80, 152-183, 222-245, 348-362 and 363
The light chain or part thereof is SEQ ID NO: 34-37, 184-221,
In the method disclosed in item 64 having a sequence selected from the group consisting of 364 and 365
is there.
Item 66. Another embodiment further comprises isolating the antibody or antigen-binding fragment thereof.
The method disclosed in item 64 or 65.
Item 67. Another embodiment is an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier
The method disclosed in any of items 64-66, further comprising preparing a composition comprising a body.
It is.
Item 68. Another embodiment is V_H-CDR1, V_H-CDR2 and V_H-Copy CDR3
A vector containing a DNA sequence to be loaded,
  V_H-CDR1 is SYGMH (SEQ ID NO: 4) or SHAMH (SEQ ID NO: 333)
The
  V_H-CDR2 is LTYHHGNRKFYADSVRG (SEQ ID NO: 5) or VIWH
DGSKKYYADSVKG (SEQ ID NO: 334),
  V_H-CDR3 is ESMGAINDN (SEQ ID NO: 6) or EWVGGTCDS (sequence
No. 335).
Item 69. Another embodiment is V_L-CDR1, V_L-CDR2 and V_L-Copy CDR3
A vector containing a DNA sequence to be loaded,
  V_L-CDR1 is IGNNNNIGSHAVG (SEQ ID NO: 7) or SGNSSNI
GYAVG (SEQ ID NO: 330),
  V_L-CDR2 is GRPSPPSG (SEQ ID NO: 8) or GKSPASG (SEQ ID NO: 3
31)
  V_L-CDR3 is STWDDSLSAVV (SEQ ID NO: 9) or STWDDSLRLSA
It is a vector that is VL (SEQ ID NO: 332).
Item 70. Another embodiment is:
  (I) contacting a biological sample or peptide library with a probe containing an epitope
Letting
  (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. In another embodiment, the molecule is an anti-hGM-CSF monoclonal antibody or
72. The method disclosed in item 70 or 71, which is an antigen-binding fragment of
Item 73. Another embodiment, the item 70-, wherein the probe further comprises a detectable marker
72. The method disclosed in any of 72.
Item 74. Another embodiment is any of items 70-73, wherein the probe is immobilized
The method disclosed in item 1.
Item 75. Another embodiment binds to hGM-CSF (hGM-CSF) and hGM-C
Anti-hGM-CSF monochrome capable of neutralizing the physiological activity of SF (hGM-CSF)
Null antibody or antigen-binding portion thereof, selected from the group consisting of SEQ ID NOs: 4-9
Having a complementarity determining region (CDR) represented by one or more amino acid sequences
An anti-hGM-CSF monoclonal antibody or an antigen-binding portion thereof.
Item 76. Another embodiment binds to hGM-CSF and neutralizes hGM-CSF bioactivity.
Anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof
One or more amino acid sequences selected from the group consisting of SEQ ID NOs: 330 to 335
Anti-hGM-CS, characterized in that it has a complementarity determining region (CDR) represented by
F monoclonal antibody or antigen-binding portion thereof.
Item 77. Another embodiment is one or more amino acids within the complementarity determining region (CDR)
Item 75, characterized in that has an amino acid sequence substituted, deleted, inserted or added
Or the anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof disclosed in item 76
It is.
Item 78. Another embodiment is a method wherein TF-1 cells proliferate upon induction of hGM-CSF.
Characterized in that it inhibits proliferation of TF-1 cells by about 50% at a concentration of about 14 pM,
75-anti-hGM-CSF monoclonal antibody disclosed in item 77 or antigen-binding portion thereof
Minutes.
Item 79. Another embodiment is characterized in that it inhibits the proliferation of peripheral blood dendritic cells,
78, an anti-hGM-CSF monoclonal antibody disclosed in 78, or an antigen-binding portion thereof.
Item 80. Another embodiment is 4 × 10 4 for hGM-CSF.^-TenK below M_DHigh affinity with value
Or an anti-hGM-CSF monoclonal antibody disclosed in Item 78,
Or an antigen-binding portion thereof.
Item 81. Another embodiment is IgG₁(Λ) belonging to class (subclass)
The anti-hGM-CSF monoclonal antibody disclosed in item 75 to item 80 or
The antigen-binding portion of
Item 82. Another embodiment is a human monoclonal antibody, item 7
The anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof disclosed in item 5 to item 81
It is.
Item 83. Another embodiment is the anti-hGM-CSF monoc disclosed in items 75-82.
HGM-CSF comprising a local antibody or antigen-binding moiety and a pharmaceutically acceptable carrier
A pharmaceutical composition for diseases caused by.
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
The medical agent disclosed in item 83, wherein the medical agent is any one selected from the group consisting of
It is a pharmaceutical composition.
Item 85. Another embodiment binds to hGM-CSF and neutralizes hGM-CSF bioactivity.
An anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof
Isolated deoxyribonucleic acid (DNA) comprising the group consisting of SEQ ID NOs: 4 to 9
An isolated amino acid sequence comprising at least one selected from
DNA.
Item 86. Another embodiment binds to hGM-CSF and neutralizes hGM-CSF bioactivity.
An anti-hGM-CSF monoclonal antibody or antigen-binding portion thereof
Isolated DNA selected from the group consisting of SEQ ID NOs: 330 to 335
Isolated DNA, characterized in that it encodes an amino acid sequence comprising at least one
is there.
Item 87. Another embodiment is the same as the DNA disclosed in item 85 or item 86 under stringent conditions
Isolated DNA that can hybridize with
Item 88. Another embodiment is the isolated D disclosed in any of items 85 through 87
It is a vector characterized by incorporating NA.
Item 89. In another embodiment, the recombinant expression vector disclosed in item 88 is introduced.
A host cell characterized by this.
Item 90. Another embodiment is a method for enhancing activity, characterized by the same specific antigen.
Multiple types of anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof that are different
It is the method characterized by administering simultaneously.
Item 91. In another embodiment, the plurality of types of anti-hGM-CSF monoclonal antibodies
Or the antigen-binding portion thereof is the following (a) and (b):
  (A) SEQ ID NOs: 4 to 9, SEQ ID NOs: 330 to 335, SEQ ID NOs: 336 to 341,
Is the complementarity determining region (CDR) represented by the amino acid sequences of SEQ ID NOs: 342 to 347
An anti-hGM-CSF monoclonal antibody having specificity for hGM-CSF or a
An antigen-binding portion,
  (B) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added;
The anti-hGM-CSF monoclonal antibody of (a) having a sequence and specific for hGM-CSF
Or its antigen-binding portion
Item comprising two or more types of antibodies or antigen-binding portions thereof selected from
90 for enhancing the activity disclosed in 90.
Item 92. Another embodiment is a method wherein TF-1 cells proliferate upon induction of hGM-CSF.
Two or more antibodies or antigen-binding portions thereof at a concentration of about 55 pM each.
In order to enhance the activity disclosed in item 91, characterized by inhibiting the growth of cysts by 80% or more
This is the method.
Item 93. Another embodiment provides for multiple types of anti-hGM-C that are specific for the same specific antigen.
A physician comprising an SF monoclonal antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier
It is a pharmaceutical composition or an animal pharmaceutical composition.
Item 94. Another embodiment provides a plurality of types of anti-hGM-CSF monoclonal antibodies or
The antigen-binding portion is represented by the following (a) and (b):
  (A) SEQ ID NOs: 4 to 9, SEQ ID NOs: 330 to 335, SEQ ID NOs: 336 to 341,
Is the complementarity determining region (CDR) represented by the amino acid sequences of SEQ ID NOs: 342 to 347
An anti-hGM-CSF monoclonal antibody having specificity for hGM-CSF or a
An antigen-binding portion,
  (B) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added;
The anti-hGM-CSF monoclonal antibody of (a) having a sequence and specific for hGM-CSF
Or its antigen-binding portion
Item comprising two or more types of antibodies or antigen-binding portions thereof selected from
93, or a pharmaceutical composition for animals.
Item 95. Another embodiment is a method wherein TF-1 cells proliferate upon induction of hGM-CSF.
The two or more antibodies or antigen-binding portions thereof at a concentration of about 55 pM each
The pharmaceutical composition disclosed in Item 94, which inhibits the growth of one cell by 80% or more.
Or a pharmaceutical composition for animals.

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 are isolated from blood of donors with high serum anti-GM-CSF monoclonal antibody titers and then E
Infected with BV. 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 using recombinant hGM-CSF (rhGM-CSF
) Was performed by ELISA using a 96-well plate coated with. Cells in wells positive for antibody production are seeded on 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 antibody against the presence of anti-hGM-CSF monoclonal antibody. Screened. Cells in wells positive for antibody production were 96
The ultradilution culture on the well plate took 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 and subclasses of 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 is transformed into P
Amplified by CR. 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 amino acid sequence of antibody based on nucleotide sequence cDNA of antibody gene [EV100 each consisting of heavy (H) chain and light (L) chain genes
7, EV1018, and EV1019] were cloned into plasmid vectors and their nucleotide sequences were analyzed by a sequencer (ABI). These amino acid sequences were determined based on the nucleotide sequences obtained. Antibody (EV1007, EV10
18, EV1019, and EV1003) complementarity determining regions (CDRs)
The abat method was used. The CDR sequences of the four antibodies are represented by SEQ ID NOs: 4-9 and 330-347. Specifically, EV1018 L chain CDR1, L chain CDR2, L chain C
DR3, H chain CDR1, H chain CDR2, and H chain CDR3 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. EV
1003 L chain CDR1, L chain CDR2, L chain CDR3, H chain CDR1, H chain CDR2,
And the heavy chain CDR3 are shown in SEQ ID NOs: 336 to 341, respectively. EV1007 L chain CDR1, L chain CDR2, L chain CDR3, H chain CDR1, H chain CDR2, and H chain CD
R3 is 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 treated with anti-G
Screening was performed for expression of the M-CSF monoclonal antibody, and a cell clone stably expressing the anti-GM-CSF monoclonal antibody was obtained.

Antibody Purification A CHO cell clone stably expressing the anti-GM-CSF monoclonal antibody was cultured in serum-free medium. After each expression period, culture supernatants are collected and antibodies are collected according to the manufacturer's instructions HiTraprProteinA FF prepack column (Amersham).
Was isolated by infinity chromatography. Purified antibody is ELI
Has binding activity to hGM-CSF by SA and about 50 by SDS-PAGE.
It was confirmed that the antibody consists of an antibody of kDA H chain and about 25 kDa L chain.

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 a method for analyzing the interaction between an antibody and an antigen, the purified antibody is captured on the sensor chip by interaction with protein G immobilized on the sensor chip surface;
The recombinant antigen was then injected into the sensor chip. Recombinant hGM-CSF using purified antibody
Was used as an antigen.
Yeast-derived (Leukine Berle) against anti-hGM-CSF monoclonal antibody
Table 2 shows the equilibrium dissociation constants (K _D ) for x) and E. coli-derived (Peprotech) recombinant hGM-CSF.

Table 2

In this assay, K _D values for yeast GM-CSF (Leukine) is, EV
It was 1.2 × 10 ⁻⁹ M for 1007, 2.3 × 10 ⁻¹⁰ M for EV1018, and 3.6 × 10 ⁻¹⁰ M for EV1019. The K _D value for the previously produced monoclonal antibody EV1003 was 2.3 × 10 ⁻¹⁰ M.
K _D values against E. coli (Peprotech) is the EV1007 9.3 × 10 ^-10 M
EV1018 is 1.5 × 10 ⁻¹⁰ M, EV1019 is 1.5 × 10 ⁻¹⁰ M, EV10
03 was 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 proliferation of peripheral blood dendritic cells 5 × 10 ⁵ dendritic cells (DC) including plasmacytoid DC and myeloid DC
Dendritic Cell Isolation Kit II Human (M
obtained from 7 × 10 ⁷ human mononuclear cells using iltenyi Biotech).
The obtained DC was 10% FCS, rhGM-CSF (1 ng / mL, Peprotech).
, TNF-α (10 ng / mL), and anti-GM-CSF monoclonal antibody (1 μg / mL)
mL) in RPMI 1640 medium (Gibco) supplemented, inoculated into 96 well flat bottom plates at a concentration of 2 × 10 ⁴ cells / well and incubated for 10 days.
Anti-GM-CSF polyclonal antibody produced by the present inventors (anti-GM-CSFpAb,
R & D) (1 μg / mL) and human anti-human cytomegalovirus monoclonal antibody (
hIgG) (1 μg / mL) was used as a control. 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 h
IgG did not inhibit.
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. Purified anti-hGM-CSF monoclonal antibody (EV
1007, EV1018, and EV1019) and the neutralizing ability of EV1003 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. G
Antibodies with M-CSF neutralizing activity were able to block the added recombinant GM-CSF activity, resulting in inhibition of GM-CSF dependent TF-1 cell growth.
4 and 5 show yeast hGM-CSF (Leukine) and E. coli hG, respectively.
It shows the inhibitory effect (neutralizing activity) of the antibody on the proliferation of TF-1 stimulated by M-CSF (Peprotech).

Each antibody was evaluated independently for its inhibitory effect, anti-GM-CSFpAb and hIgG
Was used as a control 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 assessed 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.

Yeast rhGM-CSF (Leukine) at a concentration of 0.5 ng / mL was used to stimulate the growth of TF-1 cells, and the results of the neutralizing ability of each of the anti-hGM-CSF monoclonal antibodies are shown in FIG.
Shown in 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) is 125 ng / mL or 125 ng / mL
Inhibition of cell growth depending on antibody concentration exceeding.
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. EV1018 and EV1019
Was confirmed to have a particularly high neutralizing activity against hGM-CSF. EV100
7 shows apparent 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) is 31 ng / m
Antibody concentration dependent cell growth inhibition was shown above L or 31 ng / mL.
Among the purified monoclonal antibodies, EV1003 is 31 ng / mL or 31 ng
A clear inhibition of TF1 cell proliferation was shown in a concentration-dependent manner above / mL. EV101
8 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 Cell Counting Kit (WST-1 assay,
DOJIN), 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, Mix-2: EV1003 + 1007, Mix-3: EV10
The inhibitory effects of 03 + 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: EV1
003 + 1019 increases TF1 cell growth by 10% of the positive control at each antibody concentration of 8 ng / mL
(About 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 two antibodies combined displayed particularly high neutralizing activity.
Although the inhibitory effect of EV1007 when used alone is very low, Mix-2: EV1003
+1007 shows the growth of TF1 cells at 8 ng / at each antibody concentration, as shown in FIGS.
Inhibited to 10% of the positive control in mL (approximately 55 pM).

As shown in FIG. 7, TF-1 was obtained using E. coli rhGM-CSF (Peprotech).
When cell proliferation is stimulated, cell growth is determined at each concentration of 31 ng / mL or 31 ng / m
Mix-2 exceeding L: EV1003 + 1007, Mix-3: EV1003 + 1018
, And Mix-4: EV1003 + 1019 almost completely inhibited. 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, four antibodies (EV1003, EV1007, EV101)
8 and EV1019) EV1003 showed the strongest inhibition when used alone at a concentration of 2 μg / ml, but about 20% of cells remained at this high concentration. In contrast, Mix-2, Mix-3, and Mix-4 range from 31 ng / mL to 125 ng /
Cell growth was almost completely inhibited in the concentration range up to mL. These three combinations (Mix-
2, Mix-3, and Mix-4) were pointed out to exhibit particularly high neutralizing activity.

Anti-GM-CSF monoclonal antibody (EV1003) and anti-CMV antibody (hIgG)
Mix-1, which was a combination use of, did not enhance the inhibitory effect of the antibody compared to the use of these alone.
Among the three antibodies obtained in the present invention, two antibodies (EV1018 and EV10
19) 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 resulting in various diseases, and hGM-CSF
Can be inactivated (neutralized). Thus, the antibody or antigen-binding portion thereof has a higher hG than the anti-hGM-CSF monoclonal antibody produced to date.
Has neutralizing activity against M-CSF. 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 useful for asthma, COPD, cystic fibrosis, intestinal lung disease (intes
allergic diseases such as tinal long disease), rhinitis, atopy, and hay fever, graft rejection, graft-versus-host disease (GVHD), arthritis and related joint diseases, psoriasis, myeloid leukemia, multiple sclerosis, Alzheimer It is effective as a prophylactic or therapeutic agent at low doses for diseases and diseases associated with elevated GM-CSF including rheumatoid arthritis, but the effects are 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 (anti-GM-
All CSF monoclonal antibodies recognize rhesus 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.

Mouse GM-CSF (Pepro Tech and Biomol; for example, GenBa
nk acceptance number NP — 034099) and measurements 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−ＣＳ
Ｆ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 between GM-CS between mice and humans.
F protein hybrids have been established.

Marmoset GM-C similar to rhesus monkey GM-CSF expression clone described above
An SF expression clone (eg, GenBank accession number B0KWQ4 having 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 of hGM-CSF expressed in E. coli (Fa. Biomol # 2514) by using standard techniques well known in the art.
-CSF (rhGM-CSF), or (ii) Rhesus monkey GM-CSF (e.g.
GenBank accession number NP_001028121) recombinant rhesus monkey GM-CS
F (rrGM-CSF) or (ii) Marmoset GM-CSF (eg GenBank accession number B0 with methionine at position 53 and proline at position 109)
KWQ4) (both HEK using standard techniques well known in the art)
Recombinant GM-CSF (rGM-CSF) against various species is used, such as recombinant marmoset GM-CSF (rmGM-CSF) (expressed at 293).

TF-1 cells were cultured in cell culture medium (RPMI1640 (Cat. 31870 Fa. Gib).
co), 1 × Glutamax100 × (Cat.35050-038Fa.Gibco
) 1 mM Pyrobat sodium (Sodium Pyrovat) 100 mM (Cat
. 11360-039Fa. Gibco)), 10 mM Hepes 1M (Cat. 1)
5690-056 Fa. Gibco), 10% FCS (Cat. 10500-064 Fa
. Gibco), 2 ng / ml rGM-CSF (Cat. 200-005LFa.Re
liaTech GmbH). Cells were washed 3 times with PBS, 1E5 cells /
Inoculate in 96-well plates (Cat. 167008 Fa. Nunc) in assay medium (cell culture medium without rGM-CSF) at a concentration of 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. Kit (CellTiter 96 Aqueous One
Solution Cell Proliferation Assay, Fa. Pro
mega, Cat. No. Add 20 μl MTS according to the instructions of G3581) and add 4
Cell viability is measured by measuring the absorbance at 92 nm. Negative control is rGM
-TF-1 cells grown in cell culture medium without CSF. The positive control is ED
TF-1 cells grown in cell culture medium containing 80 concentrations of rGM-CSF.

GM-CS of humans, rhesus monkeys, and marmosets before testing for neutralizing activity of antibodies
ED80 for stimulating proliferation on F was determined (Table 8). 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) is marmoset GM-CSF
Is a somewhat ineffective stimulator of TF-1 proliferation. Thus, the human cell line TF-
1 is suitable for testing neutralizing activity against human and rhesus monkey GM-CSF,
It is not suitable for testing GM-CSF derived from marmoset.

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. ED for human, rhesus monkey, and marmoset GM-CSF in TF-1 proliferation assay
80

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, EV10
An 18 heavy chain variant was produced and tested for its neutralizing activity. As shown in Table 9, the result is
It is pointed out that the bioactivity of EV1018 is maintained after removal of the N-linked glycosylation site in heavy chain framework 3. Table 9 shows WT containing N-glycosylation sites.
IC50 values are shown for two glycosylation site sequence variants (EV1018 variants 1 and 2) designated N95K and T97A, respectively, compared to the EV1018 heavy chain. Each purification I
IC50 values for gG were measured in a TF-1 assay as essentially 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 antibody, EV101
8, 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 are wild type EV10
Equally neutralizes 18 activities.

In the experimental setup that resulted in the results shown in Table 9, under a given ED80 concentration, ie r
Because reliable results cannot be obtained under a dose of GM-CSF of 1.5 ng / ml,
Commercially available rat anti-human GM-CSF IgG2aBVD2-21C11 (Fa.BD, # 5
54503) 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, various rGM- as indicated between the standard antibody and the antibody of interest.
A much more reliable comparison is possible with just the CSF dose. For example, WO2006 / 1
22797 (pages 56 and 57, Table 8) disclosed that the best antibody was 35 times better than the commercially available rat anti-human GM-CSF, clone BVD2-21C11.
Clone BVD2-21C11 and E, which are commercially available anti-human GM-CSF monoclonal antibodies
The ratio between IC50s between V1018 is 413. Therefore, based on comparison to commercial standards, the neutralizing activity of EV1018 is about 10 times higher as shown in Table 10. WO200
6/122797 indicates to those skilled in the art that the neutralizing activity of the anti-GM-CSF monoclonal antibody is GM-C
This is surprising because it teaches a strong correlation with the binding affinity for SF. (WO 2006/122797, binding affinity improvement shown in Table 4 on page 50 is 52-
Correlating with the improvement in neutralization efficacy shown in Table 6 and Table 7 on page 54).

The binding affinity of MOR04357 (WO 2006/122797, page 50, Table 4: MO
Compared to the binding affinity of EV1018 (R04357-Fab has a binding affinity of 7 pM), we understand that the binding of EV1018 to GM-CSF is about 16-fold 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 (C
at. 31870 Fa. Gibco), 1 × Glutamax, 100 × (Cat. 35)
050-038 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-
Eight cytokine levels were measured using OptEIA human IL-8 Elisa Set (Fa. BD
Bioscience, Cat. No. 555244).

The negative control is U937 cells grown in cell culture medium without addition of antibody and without addition of GM-CSF, and the positive control is ED80 rGM-CSF without addition of antibody solution.
U937 cells grown in cell culture medium containing Human and rhesus monkey GM-
ED80 concentration relative to CSF was measured (Table 9) and this concentration was used to determine various anti-GM-CS.
IC50 against F monoclonal antibody is measured (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 are human GM-CSF and rhesus monkey GM.
-Neutralized CSF very efficiently. EV1018 has a higher neutralizing activity than EV1019 and much higher neutralizing ability than EV1003. All variants of EV1018 are wild type EV
It represents a neutralizing activity equal to 1018.

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 adheres to the adhesion molecule CD11b / Mac on the surface of granulocytes.
Guide 1 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 effectiveness of anti-GM-CSF antibody was confirmed by the adhesion marker CD11b on primary h granulocytes.
Tested against GM-CSF mediated induction of / Mac1.

80 μl of coagulation-inhibited peripheral blood from a healthy donor was added to rhGM-CSF (final concentration 30 p
M) Anti-GM-C preincubated with (Fa Biomol, # 2514) for 20 minutes
SF monoclonal antibody or isotype antibody (human IgG, # I-2511; Fa. Sig
Incubate with ma-Aldrich) (positive control) at 37 ° C. for 15 minutes. 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, phycoerythrin (Fa: Pharmi)
anti-CD11b antibody labeled with ngen; 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. Erythrocyte, red bloo
d cell) is dissolved by adding 2 ml of Lysis solution (Fa: BD; Cat349202) and incubated for an additional 10 minutes at room temperature in the dark. PBS, 0.
After two washing steps with 1% BSA, the leukocytes were transferred to Cellfix (Fa: BD; Cat
340181) Suspend in 500 μl. For analysis, LSRII flow cytometer,
And DIVA 6.1.1 software for FACS data analysis (both Fa
. BD). Granulocyte cell populations are plotted in dot plots using forward and lateral scatter (d
optlot) 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 smoke such as rats and mice causes many pathophysiologically relevant anatomical lesions comparable to human disease (Fujita and Nakanishi, 2007).
Year). 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 300 μl of vehicle (PBS, 10 mM NaCl) was administered intraperitoneally (ip) 2 hours 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 by Bender MedSystems (Flow Cy
tomix). Briefly, 25 μl of BALF supernatant was converted to 25 μl of 1 × assay buffer (Basic Kit BMS8440FF), Bea
d Mixture (mixture of MCP-1 and MIP-1a, Simplex Kits,
BMS86005FF, BMS86013FF) 25 μl, biotin conjugate mixture (included in Simplex Kits) 50 μl, and incubated for 2 hours at room temperature on a 500 rpm microplate shaker. After washing twice with assay buffer, the beads are dissolved in 100 μl of assay buffer and streptavidin-
50 μl of PE-solution (included in Basic Kit) was added. 500 beads
Incubated for 1 hour at room temperature on a microplate shaker at rpm. The beads were washed twice with assay buffer. Dissolve the beads in 500 μl of assay buffer,
FlowCytomix Pr supplied by Bender MedSystems
Analyzed by flow cytometry using o2.2 software. For quantification, standard dilutions (included in the Simplex Kit) were taken from the supplier (Bender Me
dSystems). Cytokine concentrations were determined based on standards.
When mice were pretreated with anti-GM-CSF antibody, MPO activity was reduced by approximately 25% (FIG. 9).
A). 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 (irrelevant humanized IgG1, FAP-recognizing negative control antibody (US20030196868, Use of FAP alpha spe
cifi antibody body BIBH1 in the treatment of
cancer (http://www.asco.org/ASCO/Abstrac
ts + & + Virtual + Meeting / Abstracts? & Vmview = a
bst_detail_view & confID = 10 & abstractID = 102
8)), 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 several 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). PBS + 1 for 3 hours at room temperature with gentle rocking
Blocked with% BSA + 0.1% Tween20. Antibody in probing buffer (
probing buffer) (PBS, 5 mM MgCl ₂ , 0.5 mM DTT,
Dilute to 5 μg / ml in 0.05% Triton X-100, 5% glycerol, 1% BSA) and incubate on the array for 1-2 hours at 4 ° C. The array was then washed three 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. These were washed again as described above, dried by centrifugation (800 × g), Cy5 filter set and steady PMT
Scanned in a Perkin-Elmer Proscan microarray scanner.

Epitope mapping by ortholog substitution was also performed. The tested antibodies are conformational,
Or a properly folded GM-CS since it recognizes non-contiguous epitopes
It is only possible to determine each epitope in the F situation. B described above
As shown in the iacore results, the antibody in question only binds weakly, if any, to mouse GM-CSF. Therefore, the present inventors have selected human GM-CS of the selected region
A chimeric version of GM-CSF was designed in which F 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. Chimeras 3 and 6, GM-CSF 3
Spatial mapping onto the dimensional crystal structure indicates that these chimeras are adjacent to each other on one side of the GM-CSF molecule, and is in good agreement with these findings. 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 that contain mouse amino acids in the position represented by chimera 4 or 5 are E
Shows weak binding to V1019. 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. GM of chimeras 4 and 5
-Due to the spatial mapping of CSF onto the three-dimensional crystal structure, these chimeras
It is shown to be adjacent to each other on one side of the F molecule and is in good agreement with these findings. 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). 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 that contain mouse amino acids in the position represented by chimera 4 or 5 are E
Shows weak binding to V1018. 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. Due to the spatial mapping of chimeras 4 and 5 onto the three-dimensional crystal structure of GM-CSF, these chimeras are
-It is shown to be adjacent to each other on one side of the CSF molecule and is in good agreement with these findings. This surface represents a discontinuous epitope recognized by EV1018. Since both regions are required for high affinity binding of EV1018, the discontinuous epitope is
Exist close to each other and on or near the surface of GM-CSF (and thus
Must consist of amino acids from variants 4 and 5 (which affect the amino acid residues exposed on the surface). These are amino acids “ELYK” and “TMMA”
SHYKQH ".

These results show that EV1018 and EV1019 recognize the same discontinuous epitope of the 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
MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYK
QGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE
(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
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 10)

> EV1018-wt IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 11)

> EV1018-T97A IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN A LY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 12)

> EV1018-T97V IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN V LY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 13)

> EV1018-N95D IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS D NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 14)

> EV1018-N95E IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS E NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 15)

> EV1018-N95K IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS K NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 16)

> EV1018-N95Q IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS Q NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 17)

> EV1018-N93Q-N95T IgG1KO
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRD Q S T NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 18)

> EV1018-wt IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 19)

> EV1018-T97A IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN A LY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 20)

> EV1018-T97V IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN V LY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 21)

> EV1018-N95D IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS D NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 22)

> EV1018-N95E IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS E NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 23)

> EV1018-N95K IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS K NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 24)

> EV1018-N95Q IgG1-BI
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS Q NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 25)

> EV1018-N93Q-N95T IgG1-BI QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHH
GNRKFYADSVRGRFTISRD Q S T NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 26)

> EV1018-T97A IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN A LY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 27)

> EV1018-T97V IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNN V LY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 28)

> EV1018-N95D IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS D NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 29)

> EV1018-N95E IgG1-original-stationary QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLE
WVALTYHHGNRKFYADSVRGRFTISRDNS E NTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK
(SEQ ID NO: 30)

> EV1018-N95K IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS K NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 31)

> EV1018-N95Q IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNS Q NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 32)

> EV1018-N93Q-N95T IgG1-original-stationary
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRD Q S T NTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 33)

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

> EV1018-wt-BI
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQP
EDEADYYCSTWDSSLSAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 35)

> EV1018-wt-BI-G1
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQP
EDEADYYCSTWDSSLSAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 36)

> EV1018-wt-original-steady
QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQP
EDEADYYCSTWDSSLSAVVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 37)

Heavy chain without EV1018 signal sequence
> EV1018-wt-IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 38)

> EV1018-T97A-IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 39)

> EV1018-T97V-IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 40)

> EV1018-N95D-IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 41)

> EV1018-N95E IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 42)

> EV1018-N95K IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 43)

> EV1018-N95Q IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 44)

> EV1018-N93Q-N95T IgG1-KO-QVQL
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 45)

> EV1018-wt IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 46)

> EV1018-T97A IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 47)

> EV1018-T97V IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 48)

> EV1018-N95D IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 49)

> EV1018-N95E IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 50)

> EV1018-N95K IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 51)

> EV1018-N95Q IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 52)

> EV1018-N93Q-N95T IgG1-QVQL-BI
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 53)

> EV1018-IgG2
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 54)

> EV1018-wt-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 55)

> EV1018-T97A-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 56)

> EV1018-T97V-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 57)

> EV1018-N95D-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 58)

> EV1018-N95E-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 59)

> EV1018-N95K-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 60)

> EV1018-N95Q-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 61)

> EV1018-N93Q-N95T-IgG2
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 62)

> EV1018-IgG4
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 63)

> EV1018-wt-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 64)

> EV1018-T97A-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 65)

> EV1018-T97V-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 66)

> EV1018-N95D-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 67)

> EV1018-N95E-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 68)

> EV1018-N95K-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 69)

> EV1018-N95Q-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 70)

> EV1018-N93Q-N95T-IgG4
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 71)

> EV1018-IgG4-SP
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 72)

> EV1018-wt-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 73)

> EV1018-T97A-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNALY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 74)

> EV1018-T97V-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNVLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 75)

> EV1018-N95D-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSDNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 76)

> EV1018-N95E-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSENTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 77)

> EV1018-N95K-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSKNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 78)

> EV1018-N95Q-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSQNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 79)

> EV1018-N93Q-N95T-IgG4-SP
QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDQSTNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 80)

Heavy chain with EV1018 signal sequence
> EV1018
MEFGLIWVFLVTLLRGVQC QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 81)

> EV1018-wt-IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 82)

> EV1018-T97A-IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 83)

> EV1018-T97V-IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 84)

> EV1018-N95D-IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 85)

> EV1018-N95E IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 86)

> EV1018-N95K IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 87)

> EV1018-N95Q IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 88)

> EV1018-N93Q-N95T IgG1KO
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 89)

> EV1018-wt IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 90)

> EV1018-T97A IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 91)

> EV1018-T97V IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 92)

> EV1018-N95D IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 93)

> EV1018-N95E IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 94)

> EV1018-N95K IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 95)

> EV1018-N95Q IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 96)

> EV1018-N93Q-N95T IgG1-BI
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 97)

> EV1018-T97A IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 98)

> EV1018-T97V IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 99)

> EV1018-N95D IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 100)

> EV1018-N95E IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 101)

> EV1018-N95K IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 102)

> EV1018-N95Q IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 103)

> EV1018-N93Q-N95T IgG1-original-stationary
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 104)

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

> EV1018-wt-BI
MGWSCIILFLVATATGVHS QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDR
FSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 106)

> EV1018-wt-BI2 (G1) MGWSCIILFLVATATGVHS QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQIS
RGAPKMVLFGRSPPSGVPDRFSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLGQPKAAPSVTLF
PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE
GSTVEKTVAPTECS
(SEQ ID NO: 107)

> EV1018-wt-original-steady
MGWSCIILFLVATATGVHS QSALTQETSVSGTVGQKVTLSCIGNNNNIGSHAVGWYQQISRGAPKMVLFGRSPPSGVPDR
FSGSKSGTTASLIISGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 108)

Heavy chain with EV1018 signal sequence
> EV1018-wt-IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 109)

> EV1018-T97A-IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 110)

> EV1018-T97V-IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 111)

> EV1018-N95D-IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 112)

> EV1018-N95E IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 113)

> EV1018-N95K IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 114)

> EV1018-N95Q IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 115)

> EV1018-N93Q-N95T IgG1-KO-QVQL
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 116)

> EV1018-wt IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 117)

> EV1018-T97A IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 118)

> EV1018-T97V IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 119)

> EV1018-N95D IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 120)

> EV1018-N95E IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 121)

> EV1018-N95K IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 122)

> EV1018-N95Q IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 123)

> EV1018-N93Q-N95T IgG1-QVQL-BI
MGWSCIILFLVATATGVHS QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 124)

> EV1018-IgG2
MEFGLIWVFLVTLLRGVQC QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 125)

> EV1018-wt-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 126)

> EV1018-T97A-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 127)

> EV1018-T97V-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 128)

> EV1018-N95D-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 129)

> EV1018-N95E-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 130)

> EV1018-N95K-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 131)

> EV1018-N95Q-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 132)

> EV1018-N93Q-N95T-IgG2
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 133)

> EV1018-IgG4
MEFGLIWVFLVTLLRGVQC QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 134)

> EV1018-wt-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 135)

> EV1018-T97A-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 136)

> EV1018-T97V-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 137)

> EV1018-N95D-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 138)

> EV1018-N95E-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 139)

> EV1018-N95K-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 140)

> EV1018-N95Q-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 141)

> EV1018-N93Q-N95T-IgG4
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 142)

> EV1018-IgG4-SP
MEFGLIWVFLVTLLRGVQC QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 143)

> EV1018-wt-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 144)

> EV1018-T97A-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNALYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 145)

> EV1018-T97V-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSNNVLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 146)

> EV1018-N95D-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSDNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 147)

> EV1018-N95E-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSENTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 148)

> EV1018-N95K-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSKNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 149)

> EV1018-N95Q-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDNSQNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 150)

> EV1018-N93Q-N95T-IgG4-SP
MGWSCIILFLVATATGVHS QVELVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYA
DSVRGRFTISRDQSTNTLYLQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 151)

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

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

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

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

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

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

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

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

Heavy chain without EV1019 signal sequence
> EV1019
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 160)

> EV1019-wt-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 161)

> EV1019-wt-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 162)

> EV1019-C105G
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 163)

> EV1019-C105G-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 164)

> EV1019-C105G-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 165)

> EV1019-C105S
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 166)

> EV1019-C105S-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 167)

> EV1019-C105S-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 168)

> EV1019-C105A
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 169)

> EV1019-C105A-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 170)

> EV1019-C105A-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 171)

> EV1019-C105Q
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 172)

> EV1019-C105Q-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 173)

> EV1019-C105Q-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 174)

> EV1019-C105T
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 175)

> EV1019-C105T-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 176)

> EV1019-C105T-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 177)

> EV1019-C105M
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 178)

> EV1019-C105M-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 179)

> EV1019-C105M-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 180)

> EV1019-C105L
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 181)

> EV1019-C105L-IgG1-BI
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 182)

> EV1019-C105L-IgG1KO
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 183)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

> EV1019-wt-BI
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 203)

> EV1019-wt-BI2
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 204)

> EV1019-wt-original-steady
QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 205)

> EV1019-N25S
QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 206)

> EV1019-N25S-BI2
QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 207)

> EV1019-N25G
QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 208)

> EV1019-N25G-BI2
QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 209)

> EV1019-N25T
QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 210)

> EV1019-N25T-BI2
QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 211)

> EV1019-N25R
QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 212)

> EV1019-N25R-BI2
QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 213)

> EV1019-N25Q
QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 214)

> EV1019-N25Q-BI2
QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 215)

> EV1019-S27N
QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 216)

> EV1019-S27N-BI2
QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 217)

> EV1019-S27G
QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 218)

> EV1019-S27G-BI2
QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 219)

> EV1019-S27A
QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 220)

> EV1019-S27A-BI2
QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDRFSGSKSGTTASLLVSGLQP
EDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 221)

Heavy chain without EV1019 signal sequence
> EV1019-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 222)

> EV1019-wt-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 223)

> EV1019-wt-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 224)

> EV1019-C105G-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 225)

> EV1019-C105G-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 226)

> EV1019-C105G-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 227)

> EV1019-C105S-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 228)

> EV1019-C105S-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 229)

> EV1019-C105S-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 230)

> EV1019-C105A-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 231)

> EV1019-C105A-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 232)

> EV1019-C105A-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 233)

> EV1019-C105Q-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 234)

> EV1019-C105Q-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 235)

> EV1019-C105Q-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 236)

> EV1019-C105T-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 237)

> EV1019-C105T-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 238)

> EV1019-C105T-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 239)

> EV1019-C105M-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 240)

> EV1019-C105M-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 241)

> EV1019-C105M-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 242)

> EV1019-C105L-IgG2
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV
SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 243)

> EV1019-C105L-IgG4
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 244)

> EV1019-C105L-IgG4SP
QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYADSVKGRFSVSRDNSKNTLF
LQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 245)

Heavy chain with EV1019 signal sequence
> EV1019
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 246)

> EV1019-wt-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 247)

> EV1019-wt-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 248)

> EV1019-C105G
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 249)

> EV1019-C105G-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 250)

> EV1019-C105G-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 251)

> EV1019-C105S
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 252)

> EV1019-C105S-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 253)

> EV1019-C105S-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 254)

> EV1019-C105A
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 255)

> EV1019-C105A-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 256)

> EV1019-C105A-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 257)

> EV1019-C105Q
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 258)

> EV1019-C105Q-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 259)

> EV1019-C105Q-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 260)

> EV1019-C105T
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 261)

> EV1019-C105T-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 262)

> EV1019-C105T-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 263)

> EV1019-C105M
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 264)

> EV1019-C105M-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 265)

> EV1019-C105M-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 266)

> EV1019-C105L
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 267)

> EV1019-C105L-IgG1-BI
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 268)

> EV1019-C105L-IgG1KO
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 269)

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

> EV1019-wt-BI
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 271)

> EV1019-wt-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 272)

> EV1019-wt-original-steady
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 273)

> EV1019-N25S
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 274)

> EV1019-N25S-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGSSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 275)

> EV1019-N25G
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 276)

> EV1019-N25G-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGGSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 277)

> EV1019-N25T
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 278)

> EV1019-N25T-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGTSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 279)

> EV1019-N25R
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 280)

> EV1019-N25R-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGRSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 281)

> EV1019-N25Q
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 282)

> EV1019-N25Q-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGQSSNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 283)

> EV1019-S27N
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 284)

> EV1019-S27N-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSNNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 285)

> EV1019-S27G
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 286)

> EV1019-S27G-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSGNIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 287)

> EV1019-S27A
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 288)

> EV1019-S27A-BI2
MGWSCIILFLVATATGVHS QPALTQEASVSGTVGQTVTLLCSGNSANIGSYAVGWYQQISRGAPKIVVFGKSPASGIPDR
FSGSKSGTTASLLVSGLQPEDEADYYCSTWDSRLSAVLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISD
FYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 289)

Heavy chain with EV1019 signal sequence
> EV1019-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 290)

> EV1019-wt-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 291)

> EV1019-wt-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTCDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 292)

> EV1019-C105G-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 293)

> EV1019-C105G-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 294)

> EV1019-C105G-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTGDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 295)

> EV1019-C105S-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 296)

> EV1019-C105S-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 297)

> EV1019-C105S-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTSDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 298)

> EV1019-C105A-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 299)

> EV1019-C105A-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 300)

> EV1019-C105A-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTADSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 301)

> EV1019-C105Q-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 302)

> EV1019-C105Q-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 303)

> EV1019-C105Q-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTQDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 304)

> EV1019-C105T-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 305)

> EV1019-C105T-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 306)

> EV1019-C105T-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTTDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 307)

> EV1019-C105M-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 308)

> EV1019-C105M-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 309)

> EV1019-C105M-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTMDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 310)

> EV1019-C105L-IgG2
MEFGLSWVFLAALLRGVQC QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 311)

> EV1019-C105L-IgG4
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(SEQ ID NO: 312)

> EV1019-C105L-IgG4SP
MGWSCIILFLVATATGVHS QAQLVESGGGVVKPGGSLRLSCISSKFTFSSHAMHWVRQAPGKGLEWVAVIWHDGSKKYYA
DSVKGRFSVSRDNSKNTLFLQMDSLRAEDTAVYFCGKEWVGGTLDSWGQGTLVIVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG
PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(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)
MDWTWRILFLVAAATGARSLVQLVQSEAEVKKPGASVRISCRTSGYIFPTFALHWVRQAPGQSLEWVGSINTASGKTKFS
TKFQDRLTLSSNTSATTVYMDLSGLTLDDTALYYCARDRFQNIMATILDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 320)

> EV1003 light chain (κ)
METPAQLLFLLLLWLPGTTGDIVLTQSPGTLSLSPGERVTVSCRASHRVSSNYLAWYQQRPGQSPRLLIFGASNRATGVP
DRFSGSGSGTDFALTISRLEPDDFAVYYCQQYASSPVTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF
YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 321)

> EV1007 heavy chain (IgG1)
MDWTWRILFLVAAATGAPSQVHLVQSGSELKKPGASVKVSCKASGYSFSRYGIKWVRQAPGQGLEWMGWINTRSGVPAYA
QGFTGRFVFSLDTSVDTAFLEISSLKTEDTGIYYCATRPPRFYDKTEYWEDGFDVWGRGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK
(SEQ ID NO: 322)

> EV1007 light chain (λ)
MAGFPLLLTLLTHCAGSWAQSVLTQPPSASGTPGQSVNISCSGSSSNIGNSYVYWYQQLPGTAPKLLIYRNNRRPSGVPD
RFSGSKSDTSASLAISGLRSEDEADYYCATWDDSLSGRLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLIS
DFYPGAVTVAWKADGSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEMTVAPTECS
(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
QVQLVESGGGMVQPGMPLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVALTYHHGNRKFYADSVRGRFTISRDNSNNTLY
LQMTSLRAEDTAVYFCARESMGAINDNWGQGTLVIVSS
(SEQ ID NO: 348)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Claims (4)

A pharmaceutical composition for treating a disease or disorder associated with overexpression of GM-CSF comprising two or more anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof and a pharmaceutically acceptable carrier,
The two or more anti-hGM-CSF monoclonal antibody or at least one antigen binding portion thereof, comprises a light chain comprising the sequences of the heavy chain and SEQ ID NO 321 comprising the sequence of SEQ ID NO: 320,
The at least one second of the two or more anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof is the following (1) to (3):
(1) having a heavy chain sequence comprising the sequence of SEQ ID NO: 322 and a light chain sequence comprising the sequence of SEQ ID NO: 323;
(2)
(A) a heavy chain comprising a V _H -CDR1 containing sequence, a V _H -CDR2 containing sequence, and a V _H -CDR3 containing sequence, and (b) a VL-CDR1 containing sequence, a VL-CDR2 containing sequence, and a VL-CDR3 containing Comprising a light chain comprising a sequence;
(I) the V _H -CDR1-containing sequence is SYGMH (SEQ ID NO: 4);
(Ii) the V _H -CDR2 containing sequence is LTYHHGNRKFYADSVRG (SEQ ID NO: 5);
(Iii) the V _H -CDR3-containing sequence is ESMGAINDN (SEQ ID NO: 6);
(Iv) the V _L -CDR1-containing sequence is IGNNNNIGSHAVG (SEQ ID NO: 7);
(V) the V _L -CDR2 containing sequence is GRPSPPSG (SEQ ID NO: 8);
(Vi) the V _L -CDR3 containing sequence is STWDDSLSAVV (SEQ ID NO: 9), or
(3) comprising a variable region of a heavy chain selected from the group consisting of SEQ ID NOs: 152 to 159, and a variable region of a light chain selected from the group consisting of SEQ ID NOs: 184 to 201,
Either
Diseases or disorders associated with overexpression of GM-CSF are asthma, COPD, cystic fibrosis, intestinal lung disease, rhinitis, atopy, and hay fever, graft rejection, graft versus host Selected from the group consisting of disease (GVHD), arthritis and related joint diseases, psoriasis, myeloid leukemia, multiple sclerosis, Alzheimer's disease, and rheumatoid arthritis,
A pharmaceutical composition wherein the antibody or antigen-binding portion thereof binds to hGM-CSF and can neutralize hGM-CSF activity, thereby treating the disease or disorder. A kit comprising the pharmaceutical composition of claim 1 and one or more containers. The kit of claim 2 further comprising printed instructions for using the kit. Use of two or more anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof and a pharmaceutically acceptable carrier for the manufacture of the pharmaceutical composition of claim 1 .
The two or more anti-hGM-CSF monoclonal antibody or at least one antigen binding portion thereof, the light chain saw including comprising the sequence of the heavy chain and SEQ ID NO 321 comprising the sequence of SEQ ID NO: 320,
The at least one second of the two or more anti-hGM-CSF monoclonal antibodies or antigen-binding portions thereof is the following (1) to (3):
(1) having a heavy chain sequence comprising the sequence of SEQ ID NO: 322 and a light chain sequence comprising the sequence of SEQ ID NO: 323;
(2)
(A) a heavy chain comprising a V _H -CDR1 containing sequence, a V _H -CDR2 containing sequence, and a V _H -CDR3 containing sequence, and (b) a VL-CDR1 containing sequence, a VL-CDR2 containing sequence, and a VL-CDR3 containing Comprising a light chain comprising a sequence;
(I) the V _H -CDR1-containing sequence is SYGMH (SEQ ID NO: 4);
(Ii) the V _H -CDR2 containing sequence is LTYHHGNRKFYADSVRG (SEQ ID NO: 5);
(Iii) the V _H -CDR3-containing sequence is ESMGAINDN (SEQ ID NO: 6);
(Iv) the V _L -CDR1-containing sequence is IGNNNNIGSHAVG (SEQ ID NO: 7);
(V) the V _L -CDR2 containing sequence is GRPSPPSG (SEQ ID NO: 8);
(Vi) the V _L -CDR3 containing sequence is STWDDSLSAVV (SEQ ID NO: 9), or
(3) comprising a variable region of a heavy chain selected from the group consisting of SEQ ID NOs: 152 to 159, and a variable region of a light chain selected from the group consisting of SEQ ID NOs: 184 to 201,
Is one of the uses.

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