RU2773317C2 - Antibody agents specific to human cd19, and their applications - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to an antibody or its antigen-binding fragment that specifically bind human CD19. A chimeric antigen receptor containing the specified antibody; a nucleic acid molecule encoding the specified antibody; a cell containing the specified nucleic acid molecule are also disclosed. A method for the treatment of a medical condition characterized by CD19 expression, using the specified antibody; a method for the control of T-cells to destroy target cells expressing CD19 are disclosed.

EFFECT: invention allows for the effective treatment of a medical condition characterized by CD19 expression.

32 cl, 6 ex, 9 tbl, 15 dwg

Description

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Application No. 62/240624, filed October 13, 2015, which is incorporated herein by reference in its entirety.

Sequence listing

In the present description, reference is made to a list of sequences provided electronically in the form of a .txt file with the name "ETI-2015-11_ST25.txt". The .txt file was created on October 5, 2016 and is 178,524 bytes in size. All contents of the sequence listing are incorporated herein by reference.

Prior Art

With the advent of antibody-based technologies, the development of new antibody-based therapeutics for the treatment of cancer has generated tremendous interest over the past decade. Indeed, various formats continue to be developed (eg, chimeric, humanized, human, radiolabeled, drug-conjugated, multispecific, etc.) and some have shown significant promise in the treatment of cancer. The 2015 listing reported that nearly two dozen therapeutic antibody agents were marketed or reviewed in the United States or Europe for various cancer indications (see Janice M. Reichert, PhD, Reichert Biotechnology Consulting LLC, May 26, 2015) . However, the development of particularly effective antibody agents remains a challenge.

The essence of the invention

The present invention relates, inter alia, to human antibody agents, multispecific binding agents (eg, bispecific antibodies), and chimeric antigen receptors that specifically bind CD19, in particular human CD19. In some embodiments, provided that the human antibody agents, multispecific binding agents, and chimeric antigen receptors show high specificity for human CD19 in native format (e.g., expressed on the cell surface) compared to one or more human antibody control agents , multispecific binding agents and/or chimeric antigen receptors. In some embodiments, the proposed human antibody agents, multispecific binding agents (e.g., bispecific antibodies), and chimeric antigen receptors overcome the suboptimal selectivity associated with anti-CD19 antibody-based therapeutics developed using known technologies (e.g., engineered animals, non-human, phage display, humanized antibodies derived from antibodies of mouse origin, etc.). In some embodiments, the provided human antibody agents, multispecific binding agents (eg, bispecific antibodies), and chimeric antigen receptors effectively mediate the killing of CD19-expressing cancer cells (eg, lymphomas and/or leukemias).

While embodiments using antibody agents that include human sequences (i.e., human heavy and light chain variable region sequences, including human CDR sequences) are discussed in detail herein, the present invention also relates to non-human antibody agents. In some embodiments, non-human antibody agents include human CDR sequences from the antibody agent, as described herein, and non-human framework sequences. Non-human framework sequences include, in some embodiments, any sequence that can be used to generate synthetic heavy and/or light chain variable regions using one or more human CDR sequences as described herein, including, for example, mammalian sequences, for example mice, rats, rabbits, pigs, cattle (eg cows, ox, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (eg marmosets, rhesus monkeys), etc. In some embodiments, the antibody agent of the invention comprises an antibody agent produced by grafting one or more human CDR sequences as described herein into a non-human backbone sequence (eg, a mouse or chicken backbone sequence). In many embodiments, the antibody agents provided are human antibody-based agents (e.g., a human monoclonal antibody or fragment thereof, a human antigen-binding protein or polypeptide, a human multispecific binding agent [e.g., a human bispecific antibody], a human polypeptide having one or more structural components of an immunoglobulin polypeptide person).

In some embodiments, the present invention relates to an antibody agent that specifically binds human CD19, wherein the antibody agent comprises a heavy chain CDR1 (HC-CDR1), a heavy chain CDR2 (HC-CDR2) and a heavy chain CDR3 (HC-CDR3), a light chain CDR1 (LC-CDR1), light chain CDR2 (LC-CDR2) and light chain CDR3 (LC-CDR3),

where HC-CDR1 includes GX ₁ -X ₂ -fx ₃ -SX ₄ -X ₅ (SEQ ID NO: 291), and where X ₁ is F, G, Y or V, X ₂ is S or T, X ₃ is S or T, X ₄ is N or Y and X ₅ is A, W or Y;

where HC-CDR2 includes IX ₆ -PX ₇ -X ₈ -X ₉ -X ₁₀ -T (SEQ ID NO: 292) and where X ₆ is S, D or Y, X ₇ is E, S, G or I, X ₈ is D, F or V, X ₉ is G or S and X ₁₀ is K, E, Y, D or T;

where LC-CDR1 includes SSNX ₁₁ -GX ₁₂ -X ₁₃ -X ₁₄ (SEQ ID NO: 293) or NIGSX ₁₅ -S (SEQ ID NO: 294), and where X ₁₁ is I or V, X ₁₂ is N, S or T, X ₁₃ is N, H or K, X ₁₄ is Y, A or T and X ₁₅ is K or E; and/or

where LC-CDR2 includes X ₁₆ -X ₁₇ -X ₁₈ (SEQ ID NO: 295) and where X ₁₆ is D, E, S, R or Y, X ₁₇ is N or D, and X ₁₈ is N , Y, S or D.

In some embodiments, the present invention provides a human antibody agent that specifically binds human CD19, wherein the human antibody agent comprises one or more heavy chain CDRs each having a sequence that is at least 50% identical ( for example, at least about 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of one or more CDRs heavy chain selected from Table 2, and/or one or more light chain CDRs, each having a sequence that is at least 50% identical (e.g., at least about 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98% or 99%), one or more light chain CDRs selected from Table 3.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75 and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, set forth in SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78 and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81 and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 187, SEQ ID NO: 188 and SEQ ID NO: 189.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84 and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 shown in SEQ ID NO: 190, SEQ ID NO: 191 and SEQ ID NO: 192.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87 and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 193, SEQ ID NO: 194 and SEQ ID NO: 195.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90 and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93 and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 199, SEQ ID NO: 200 and SEQ ID NO 201.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96 and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99 and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO: 210.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 211, SEQ ID NO: 212 and SEQ ID NO: 213.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 214, SEQ ID NO: 215 and SEQ ID NO: 216.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111 and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 shown in SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114 and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each CDR1 light chain, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 220, SEQ ID NO: 221 and SEQ ID NO: 222.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 223, SEQ ID NO: 224 and SEQ ID NO: 225.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120 and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 226, SEQ ID NO: 227 and SEQ ID NO: 228.

In some embodiments, the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each of which has a sequence that is at least 95% identical to a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, shown in SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, and light chain CDR1, light chain CDR2, and light chain CDR3, each having a sequence that is at least 95% identical to each of Light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NO: 229, SEQ ID NO: 230 and SEQ ID NO: 231.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, and the light chain CDRs of SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO: 183.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, and the light chain CDRs of SEQ ID NO: 184, SEQ ID NO: 185, and SEQ ID NO: 186 .

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, and the light chain CDRs of SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO: 189 .

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, and the light chain CDRs of SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192 .

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, and the light chain CDRs of SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, and the light chain CDRs of SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO: 198 .

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, and the light chain CDRs of SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201 .

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, and the light chain CDRs of SEQ ID NO: 202, SEQ ID NO: 203, and SEQ ID NO: 204.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, and the light chain CDRs of SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, and the light chain CDRs of SEQ ID NO: 208, SEQ ID NO: 209, and SEQ ID NO: 210.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, and the light chain CDRs of SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO: 213.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, and the light chain CDRs of SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, and the light chain CDRs of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, and the light chain CDRs of SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO: 222.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, and the light chain CDRs of SEQ ID NO: 223, SEQ ID NO: 224, and SEQ ID NO: 225.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, and the light chain CDRs of SEQ ID NO: 226, SEQ ID NO: 227, and SEQ ID NO: 228.

In some embodiments, the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, and the light chain CDRs of SEQ ID NO: 229, SEQ ID NO: 230, and SEQ ID NO: 231.

In some embodiments, the present invention relates to a human antibody agent that competes for CD19 binding with a human antibody agent that comprises (a) a heavy chain CDR of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75 , and light chain CDRs of SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO: 183; (b) heavy chain CDRs of SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78 and light chain CDRs of SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186; (c) heavy chain CDRs of SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81 and light chain CDRs of SEQ ID NO: 187, SEQ ID NO: 188 and SEQ ID NO: 189; (d) heavy chain CDRs of SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84 and light chain CDRs of SEQ ID NO: 190, SEQ ID NO: 191 and SEQ ID NO: 192; (e) heavy chain CDRs of SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87 and light chain CDRs of SEQ ID NO: 193, SEQ ID NO: 194 and SEQ ID NO: 195; (f) heavy chain CDRs of SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90 and light chain CDRs of SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198; (g) heavy chain CDRs of SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, and light chain CDRs of SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201; (h) heavy chain CDRs of SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96 and light chain CDRs of SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204; (i) heavy chain CDRs of SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99 and light chain CDRs of SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207; (j) heavy chain CDRs of SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102 and light chain CDRs of SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO: 210; (k) heavy chain CDRs of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, and light chain CDRs of SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO: 213; (l) heavy chain CDRs of SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108 and light chain CDRs of SEQ ID NO: 214, SEQ ID NO: 215 and SEQ ID NO: 216; (m) heavy chain CDRs of SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111 and light chain CDRs of SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219; (n) heavy chain CDRs of SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, and light chain CDRs of SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO: 222; (o) heavy chain CDRs of SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, and light chain CDRs of SEQ ID NO: 223, SEQ ID NO: 224, and SEQ ID NO: 225; (p) heavy chain CDRs of SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120 and light chain CDRs of SEQ ID NO: 226, SEQ ID NO: 227 and SEQ ID NO: 228; or (q) heavy chain CDRs of SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, and light chain CDRs of SEQ ID NO: 229, SEQ ID NO: 230, and SEQ ID NO: 231.

In some embodiments, the human antibody agent further comprises one or more amino acid substitutions in the heavy chain CDR and/or light chain CDR.

In some embodiments, the human antibody agent further comprises at least one or up to five amino acid substitutions in a heavy chain CDR and/or a light chain CDR.

In some embodiments, the human antibody agent further comprises at least one or up to three amino acid substitutions in a heavy chain CDR and/or a light chain CDR.

In some embodiments, the human antibody agent includes one or more amino acid substitutions in the heavy chain backbone and/or in the light chain structure. In certain specific embodiments, the human antibody agent comprises 1-5 amino acid substitutions in the heavy chain backbone and/or in the light chain structure.

In some specific embodiments, one or more amino acid substitutions (e.g., 1-5 amino acid substitutions) are in the framework region (i.e., heavy and/or light chain framework) and/or CDRs (i.e., heavy and/or light CDRs). chains).

In some embodiments, the human antibody agent includes one or more of the amino acid substitutions shown in Table 8. In some embodiments, the human antibody agent includes the amino acid substitution(s) found in the clone variant shown in Table 8.

In some embodiments, the present invention provides a human antibody agent that specifically binds human CD19, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 50% (e.g., at least , about 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) is identical to the heavy chain variable region sequence, which is presented in Table 1, and a light chain variable region having a sequence that is at least 50% (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the light chain variable region sequence shown in Table 1.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 2 and a light chain variable region having the sequence which is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 2.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 4 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 4.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 6 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 6.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 8 and a light chain variable region having the sequence , which is at least 95% identical to the light chain variable region sequence as shown in SEQ ID NO: 8.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 10 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 10.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 12 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 12.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 14 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 14.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 16 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 16.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 18 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 18.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 20 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 20.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 22 and a light chain variable region having the sequence , which is at least 95% identical to the light chain variable region sequence as shown in SEQ ID NO: 22.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 24 and a light chain variable region having the sequence , which is at least 95% identical to the light chain variable region sequence as shown in SEQ ID NO: 24.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 26 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 26.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 28 and a light chain variable region having the sequence , which is at least 95% identical to the light chain variable region sequence as shown in SEQ ID NO: 28.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 30 and a light chain variable region having the sequence , which is at least 95% identical to the light chain variable region sequence as shown in SEQ ID NO: 30.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 32 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 32.

In some embodiments, the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 34 and a light chain variable region having the sequence , which is at least 95% identical to the sequence of the variable region of the light chain, which is presented in SEQ ID NO: 34.

In some embodiments, the human antibody agent comprises heavy and light chain variable region sequences as set forth in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 and SEQ ID NO:34.

In some embodiments, the present invention provides a human antibody agent that specifically binds human CD19, wherein the human antibody agent comprises one or more amino acid substitutions compared to a parent (or reference) human antibody agent, and wherein the human antibody agent human antibodies include (a) heavy chain CDRs of SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126 and light chain CDRs of SEQ ID NO: 232, SEQ ID NO: 233 and SEQ ID NO: 234; (b) heavy chain CDRs of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, and light chain CDRs of SEQ ID NO: 235, SEQ ID NO: 236, and SEQ ID NO: 237; (c) heavy chain CDRs of SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132 and light chain CDRs of SEQ ID NO: 238, SEQ ID NO: 239 and SEQ ID NO: 240; (d) heavy chain CDRs of SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135 and light chain CDRs of SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 243; (e) heavy chain CDRs of SEQ ID NO: 136, SEQ ID NO: 137 and SEQ ID NO: 138 and light chain CDRs of SEQ ID NO: 244, SEQ ID NO: 245 and SEQ ID NO: 246; (f) heavy chain CDRs of SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141 and light chain CDRs of SEQ ID NO: 247, SEQ ID NO: 248 and SEQ ID NO: 249; (g) heavy chain CDRs of SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144, and light chain CDRs of SEQ ID NO: 250, SEQ ID NO: 251, and SEQ ID NO: 252; (h) heavy chain CDRs of SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 147 and light chain CDRs of SEQ ID NO: 253, SEQ ID NO: 254 and SEQ ID NO: 255; (i) heavy chain CDRs of SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150, and light chain CDRs of SEQ ID NO: 256, SEQ ID NO: 257, and SEQ ID NO: 258; (j) heavy chain CDRs of SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153 and light chain CDRs of SEQ ID NO: 259, SEQ ID NO: 260 and SEQ ID NO: 261; (k) heavy chain CDRs of SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156 and light chain CDRs of SEQ ID NO: 262, SEQ ID NO: 263 and SEQ ID NO: 264; (l) heavy chain CDRs of SEQ ID NO: 157, SEQ ID NO: 158 and SEQ ID NO: 159 and light chain CDRs of SEQ ID NO: 265, SEQ ID NO: 266 and SEQ ID NO: 267; (m) heavy chain CDRs of SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 163 and light chain CDRs of SEQ ID NO: 268, SEQ ID NO: 269 and SEQ ID NO: 270; (n) heavy chain CDRs of SEQ ID NO: 163, SEQ ID NO: 164, and SEQ ID NO: 165, and light chain CDRs of SEQ ID NO: 271, SEQ ID NO: 272, and SEQ ID NO: 273; (o) heavy chain CDRs of SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, and light chain CDRs of SEQ ID NO: 274, SEQ ID NO: 275, and SEQ ID NO: 276; (p) heavy chain CDRs of SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 171 and light chain CDRs of SEQ ID NO: 277, SEQ ID NO: 278 and SEQ ID NO: 279; (q) heavy chain CDRs of SEQ ID NO: 172, SEQ ID NO: 173 and SEQ ID NO: 174 and light chain CDRs of SEQ ID NO: 280, SEQ ID NO: 281 and SEQ ID NO: 282; (r) heavy chain CDRs of SEQ ID NO: 175, SEQ ID NO: 176, and SEQ ID NO: 177, and light chain CDRs of SEQ ID NO: 283, SEQ ID NO: 284, and SEQ ID NO: 285; or (s) the heavy chain CDRs of SEQ ID NO: 178, SEQ ID NO: 179, and SEQ ID NO: 180, and the light chain CDRs of SEQ ID NO: 286, SEQ ID NO: 287, and SEQ ID NO: 288. In certain certain embodiments, the antibody agent as described herein comprises 1-5 amino acid substitutions compared to the parent (or reference) antibody as described herein.

In some embodiments, the human antibody agent includes a light chain variable region that includes one or more amino acid substitutions at any of the amino acid positions selected from the group consisting of 10, 16, 25, 34, 52, 54, 68, 69, 72 , 75, 93, 95 and combinations thereof. In certain specific embodiments, one or more amino acid substitutions include V10M, K16E, S25N, V34I, D52N, L54Q, N68T, T69M, L72M, S75N, S93T, D95E, D95G, or a combination thereof.

In some embodiments, the human antibody agent includes a heavy chain variable region that includes one or more amino acid substitutions at any of the amino acid positions selected from the group consisting of 3, 12, 16, 17, 25, 26, 32, 63, 69 , 97, 102, 106, 108, 109, 113, 116, 117 and combinations thereof. In certain specific embodiments, one or more amino acid substitutions include Q3R, K12E, E16G, S17F, S25A, G26A, Y32F, S63F, T69A, A97V, T102S, M106L, Y108N, D109E, Q113L, L116M, M117L, or a combination thereof.

In some embodiments, the human antibody agent comprises a light chain variable region with the amino acid substitution S75N. In some embodiments, the human antibody agent comprises a light chain variable region with the amino acid substitution T69M. In some embodiments, the human antibody agent comprises a light chain variable region with amino acid substitutions D52N and D95E. In some embodiments, the human antibody agent comprises a light chain variable region with the amino acid substitutions V10M and D95G. In some embodiments, the human antibody agent comprises a light chain variable region with amino acid substitution S93T and a heavy chain variable region with amino acid substitutions S17F and T69A. In some embodiments, the human antibody agent comprises a light chain variable region with amino acid substitution N68T and a heavy chain variable region with amino acid substitutions S17F and Y108N. In some embodiments, the human antibody agent comprises a heavy chain variable region with the amino acid substitution S63F. In some embodiments, the human antibody agent comprises a heavy chain variable region with the amino acid substitutions Q3R, Y32F, and A97V. In some embodiments, the human antibody agent comprises a light chain variable region with the amino acid substitution K16E. In some embodiments, the human antibody agent comprises a heavy chain variable region with the amino acid substitution M117L.

In some embodiments, the human antibody agent comprises a light chain variable region with amino acid substitution L72M and a heavy chain variable region with amino acid substitution S25A. In some embodiments, the human antibody agent comprises a heavy chain variable region with amino acid substitutions T102S and L116M. In some embodiments, the human antibody agent comprises a light chain variable region with amino acid substitutions S25N and V34I and a heavy chain variable region with amino acid substitutions K12E and D109E. In some embodiments, the human antibody agent comprises a heavy chain variable region with the amino acid substitution E16G. In some embodiments, the human antibody agent comprises a light chain variable region with amino acid substitution L54Q and a heavy chain variable region with amino acid substitution M106L. In some embodiments, the human antibody agent comprises a light chain variable region with the S25N amino acid substitution. In some embodiments, the human antibody agent comprises a heavy chain variable region with amino acid substitutions G26A and Q113L. In some embodiments, the human antibody agent comprises a light chain variable region with the amino acid substitution L54Q. In some embodiments, the human antibody agent comprises a heavy chain variable region with the amino acid substitution T102S.

In some embodiments, amino acid substitutions are selected from Table 8.

In some embodiments, the present invention provides a human antibody agent that specifically binds human CD19, wherein the human antibody agent comprises one or more amino acid substitutions (e.g., 1-5 amino acid substitutions) compared to the parent (or reference) agent by wherein the human antibody agent comprises the heavy and light chain variable region sequences as set forth in any of SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72. In certain specific embodiments, the parent (or reference) antibody agent comprises HCVR and LCVR from SEQ ID NO: 30 or SEQ ID NO: 32.

In some embodiments, the human antibody agent is a human monoclonal antibody or a fragment thereof.

In some embodiments, the human antibody agent is a human monoclonal antibody that includes a variant Fc region. In some embodiments, the variant Fc region includes one or more amino acid substitutions compared to the original (or reference) Fc region. In some embodiments, the human monoclonal antibody includes a glycosylation variant compared to the parental human monoclonal antibody that includes a wild type (or parent or reference) Fc region. In some embodiments, the human monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In certain specific embodiments, the human monoclonal antibody is IgG1.

In some embodiments, the human antibody agent is conjugated to a therapeutic agent or detection agent.

In some embodiments, the human antibody agent is conjugated to a cytotoxic agent or fragment.

In some embodiments, the human antibody agent is conjugated to a radioisotope.

In some embodiments, the human antibody agent is or includes an immunoglobulin, Fab, F(ab') ₂ , Fd, Fv, single chain Fv (scFv), or dAb. In certain specific embodiments, the human antibody agent is a scFv. In some embodiments, the scFv includes a linker sequence. In some embodiments, the scFv is conjugated to a therapeutic agent or a detection agent. In some embodiments, the scFv is part of a chimeric antigen receptor.

In some embodiments, the present invention provides a human antibody agent as described herein for use in therapy or diagnostics.

In some embodiments, the present invention provides a human antibody agent as described herein for use in the treatment, prevention, or amelioration of a disease characterized by CD19 expression.

In some embodiments, the present invention provides a human antibody agent as described herein for use in the treatment, prevention, or alleviation of cancer.

In some embodiments, the present invention relates to an antibody-drug conjugate comprising an antigen-binding site (or antigen-binding sites) of a human antibody agent as described herein and a cytotoxic agent or group.

In some embodiments, the cytotoxic agent or fragment is or includes monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), or maytansine.

In some embodiments, the present invention provides a bispecific antibody comprising a first antigen-binding site from (or based on) a human antibody agent as described herein and a second antigen-binding site.

In some embodiments, the first and/or second antigen binding sites are selected from the group consisting of an immunoglobulin molecule, scFv, scFab, Fab, Fv, and combinations thereof. In some embodiments, the first and second antigen binding sites are configured such that they form a single polypeptide chain. In some embodiments, the first and second antigen binding sites are scFv. In some embodiments, the first and second antigen binding sites are linked by a peptide linker. In some embodiments, the second antigen binding site is linked to the C-terminus of the first antigen binding site. In some embodiments, the second antigen-binding site is linked to the N-terminus of the first antigen-binding site.

In some embodiments, the first antigen-binding site consists of an immunoglobulin molecule and the second antigen-binding site consists of scFv, scFab, Fab, or Fv.

In some embodiments, the second antigen binding site binds an immune cell selected from the group consisting of T cells, NK cells, B cells, dendritic cells, monocytes, macrophages, neutrophils, mesenchymal stem cells, and neural stem cells. In some embodiments, the second antigen binding site binds CD3 on T cells.

In some embodiments, the present invention provides a bispecific T-cell monoclonal antibody comprising the antigen-binding site of a human antibody agent as described herein.

In some embodiments, the present invention provides a bispecific antibody consisting of a first scFv that specifically binds CD19 and a second scFv that specifically binds CD3 on T cells, the first scFv comprising any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.

In some embodiments, the N-terminus of the first scFv is linked to the C-terminus of the second scFv. In some embodiments, the C-terminus of the first scFv is linked to the N-terminus of the second scFv.

In some embodiments, the N-terminus of the first scFv is linked to the C-terminus of the second scFv via a linker sequence. In some embodiments, the C-terminus of the first scFv is linked to the N-terminus of the second scFv via a linker sequence.

In some embodiments, the present invention provides a bispecific antibody as described herein for use in therapy or diagnostics.

In some embodiments, the present invention provides a bispecific antibody as described herein for use in the treatment, prevention, or amelioration of a disease characterized by CD19 expression.

In some embodiments, the present invention provides a chimeric antigen receptor comprising the antigen-binding site of a human antibody agent as described herein. In some embodiments, the chimeric antigen receptor comprises the antigen-binding site of a human antibody agent as described herein and further comprises a transmembrane domain and/or intracellular signaling domain of the native cell receptor. In certain specific embodiments, the native cell receptor is a T cell receptor (TCR). In some embodiments, the chimeric antigen receptor comprises an antigen-binding site of a human antibody agent as described herein, and further comprises a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain includes a CD3 intracellular signal sequence (e.g., CD3ξ) and an intracellular CD28 signal sequence .

In some embodiments, the antigen-binding site of the chimeric antigen receptor is scFv.

In some embodiments, the antigen-binding site of a chimeric antigen receptor as described herein is a V _L region. In certain specific embodiments, the V _L region comprises the light chain CDRs of (a) SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO: 183; (b) SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186; (c) SEQ ID NO: 187, SEQ ID NO: 188 and SEQ ID NO: 189; (d) SEQ ID NO: 190, SEQ ID NO: 191 and SEQ ID NO: 192; (e) SEQ ID NO: 193, SEQ ID NO: 194 and SEQ ID NO: 195; (f) SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198; (g) SEQ ID NO: 199, SEQ ID NO: 200 and SEQ ID NO 201; (h) SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204; (i) SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207; (j) SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO: 210; (k) SEQ ID NO: 211, SEQ ID NO: 212 and SEQ ID NO: 213; (1) SEQ ID NO: 214, SEQ ID NO: 215 and SEQ ID NO: 216; (m) SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219; (n) SEQ ID NO: 220, SEQ ID NO: 221 and SEQ ID NO: 222; (o) SEQ ID NO: 223, SEQ ID NO: 224 and SEQ ID NO: 225; (p) SEQ ID NO: 226, SEQ ID NO: 227 and SEQ ID NO: 228; (q) SEQ ID NO: 229, SEQ ID NO: 230 and SEQ ID NO: 231; (r) SEQ ID NO: 232, SEQ ID NO: 233 and SEQ ID NO: 234; (s) SEQ ID NO: 235, SEQ ID NO: 236 and SEQ ID NO: 237; (t) SEQ ID NO: 238, SEQ ID NO: 239 and SEQ ID NO: 240; (u) SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 243; (v) SEQ ID NO: 244, SEQ ID NO: 245 and SEQ ID NO: 246; (w) SEQ ID NO: 247, SEQ ID NO: 248 and SEQ ID NO: 249; (x) SEQ ID NO: 250, SEQ ID NO: 251 and SEQ ID NO: 252; (y) SEQ ID NO: 253, SEQ ID NO: 254 and SEQ ID NO: 255; (z) SEQ ID NO: 256, SEQ ID NO: 257 and SEQ ID NO: 258; (ab) SEQ ID NO: 259, SEQ ID NO: 260 and SEQ ID NO: 261; (AC) SEQ ID NO: 262, SEQ ID NO: 263 and SEQ ID NO: 264; (ad) SEQ ID NO: 265, SEQ ID NO: 266 and SEQ ID NO: 267; (ae) SEQ ID NO: 268, SEQ ID NO: 269 and SEQ ID NO: 270; (af) SEQ ID NO: 271, SEQ ID NO: 272 and SEQ ID NO: 273; (ag) SEQ ID NO: 274, SEQ ID NO: 275 and SEQ ID NO: 276; (a) SEQ ID NO: 277, SEQ ID NO: 278 and SEQ ID NO: 279; (ai) SEQ ID NO: 280, SEQ ID NO: 281 and SEQ ID NO: 282; (aj) SEQ ID NO: 283, SEQ ID NO: 284 and SEQ ID NO: 285; or (ak) SEQ ID NO: 286, SEQ ID NO: 287, and SEQ ID NO: 288. In some specific embodiments, the V _L region includes a sequence that is shown in any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO : 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 , SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO : 72.

In some embodiments, the antigen-binding site of a chimeric receptor as described herein is a V _H region. In certain specific embodiments, the V _H region comprises a heavy chain CDR of (a) SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75; (b) SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78; (c) SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81; (d) SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84; (e) SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87; (f) SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90; (g) SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93; (h) SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96; (i) SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99; (j) SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102; (k) SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105; (1) SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108; (m) SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111; (n) SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114; (o) SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117; (p) SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120; (q) SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123; (r) SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126; (s) SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129; (t) SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132; (u) SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135; (v) SEQ ID NO: 136, SEQ ID NO: 137 and SEQ ID NO: 138; (w) SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141; (x) SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144; (y) SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 147; (z) SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150; (ab) SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153; (AC) SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156; (ad) SEQ ID NO: 157, SEQ ID NO: 158 and SEQ ID NO: 159; (ae) SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 163; (af) SEQ ID NO: 163, SEQ ID NO: 164 and SEQ ID NO: 165; (ag) SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168; (a) SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 171; (ai) SEQ ID NO: 172, SEQ ID NO: 173 and SEQ ID NO: 174; (aj) SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177; or (ak) SEQ ID NO: 178, SEQ ID NO: 179, and SEQ ID NO: 180. In some specific embodiments, the V _H region includes a sequence that is shown in any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO : 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 , SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO : 72.

In some embodiments, the present invention provides a chimeric antigen receptor as described herein for use in therapy or diagnostics.

In some embodiments, the present invention provides a chimeric antigen receptor, as described herein, for use in the treatment, prevention, or alleviation of a disease characterized by CD19 expression.

In some embodiments, the present invention provides an immune effector cell that expresses a chimeric antigen receptor (or antibody agent or bispecific antibody) as described herein. In some embodiments, the immune effector cell is a T cell (eg, a cytotoxic T cell, a helper T cell, or a natural killer T cell) or an NK cell.

In some embodiments, the present invention provides an immune effector cell as described herein for use in therapy or diagnosis.

In some embodiments, the present invention provides an immune effector cell, as described herein, for use in the treatment, prevention, or amelioration of a disease characterized by CD19 expression.

In some embodiments, the present invention provides an isolated nucleic acid molecule encoding (or which encodes), in whole or in part, a human antibody agent, a bispecific antibody polypeptide chain, or a bispecific antibody or chimeric antigen receptor as described herein. In some embodiments, the isolated nucleic acid sequence includes a codon optimized sequence.

In some embodiments, the isolated nucleic acid sequence is one that is shown in any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO : 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO : 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69 and SEQ ID NO: 71.

In some embodiments, the present invention provides a vector containing an isolated nucleic acid molecule as described herein. In some embodiments, the vector is a recombinant, expression, lentiviral, or retroviral vector.

In some embodiments, the present invention provides a cell containing a vector or nucleic acid molecule, or expressing an antibody agent, a bispecific antibody, or a chimeric antigen receptor, as described herein. In some embodiments, the cell is selected from a bacterial, yeast, insect, or mammalian cell. In some embodiments, the cell is a mammalian lymphocyte (eg, a human lymphocyte).

In some embodiments, the present invention relates to a kit containing a human antibody agent, a bispecific antibody, a chimeric antigen receptor, an isolated nucleic acid molecule, a vector, or a cell as described herein.

In some embodiments, the present invention provides a kit for diagnosing a subject suffering from or predisposing to CD19 disease, or for providing a prediction of the subject's condition, the kit including a detection means for determining the concentration of CD19 present in a sample from the test subject, wherein the detection means comprises an antigen-binding site of a human antibody-based agent as described herein, a bispecific binding agent as described herein, a chimeric antigen receptor as described herein, or an immune effector cell as described herein, each of which is optional derivatized, wherein the presence of CD19 in the sample indicates that the subject is suffering from a disease associated with CD19.

In some embodiments, the present invention provides a vaccine containing an isolated nucleic acid molecule as described herein.

In some embodiments, the present invention provides a composition comprising a human antibody agent, a bispecific antibody, or a chimeric antigen receptor as described herein. In certain specific embodiments, a human antibody agent or a bispecific antibody of the composition described herein is conjugated to a cytotoxic agent or moiety.

In some embodiments, the present invention provides a pharmaceutical composition comprising a human antibody agent, a bispecific antibody, a chimeric antigen receptor, or an immune cell (or population thereof) expressing the chimeric antigen receptor as described herein, and further comprising a pharmaceutically acceptable carrier or diluent.

In some embodiments, the present invention relates to a method for producing an agent based on a human antibody, bispecific antibody or chimeric antigen receptor (or a cell expressing a chimeric antigen receptor) as described herein, including the step of culturing the cell, as described herein, in culture media under conditions allowing expression of the human antibody, bispecific antibody, or chimeric antigen receptor agent (or cell expressing the chimeric antigen receptor), and separation of the human antibody, bispecific antibody, or chimeric antigen receptor agent (or cell expressing the chimeric antigen receptor ) from the culture medium.

In some embodiments, the present invention relates to a method of treating a medical condition characterized by the expression of CD19 in a subject, comprising the step of administering a therapeutically effective amount of an agent based on a human antibody, a bispecific antibody, a chimeric antigen receptor (or an immune effector cell expressing a chimeric antigen receptor), a nucleic molecule acid or vector, as described in this document, the specified object.

In some embodiments, the medical condition characterized by CD19 expression is B-cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt's lymphoma, non-Hodgkin's lymphoma, or acute myeloid leukemia.

In some embodiments, the medical condition characterized by CD19 expression is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), diabetes, or scleroderma.

In some embodiments, the present invention relates to a method of treating cancer, the method comprising the step of administering to the subject an agent based on a human antibody, a bispecific antibody, a chimeric antigen receptor (or an immune effector cell expressing a chimeric antigen receptor), nucleotide molecules or a vector, as described herein. document.

In some embodiments, the present invention provides a method for inhibiting tumor growth, the method comprising the step of contacting tumor cells with a bispecific antibody (or human antibody agent as described herein, a chimeric antigen receptor as described herein, or an immune effector cell , as described herein, a nucleic acid molecule as described herein, or a vector as described herein), wherein the bispecific antibody consists of a first antigen-binding site based on a human antibody agent, as described herein, and a second antigen-binding site that binds the immune cell, where the contact is carried out under conditions and for a time sufficient to ensure that the destruction of tumor cells is observed (or that the immune cells to which the bispecific antibody binds inhibit the growth of tumor cells).

In some embodiments, the present invention provides a method for killing tumor cells, the method comprising the step of contacting the tumor cells with a bispecific antibody (or human antibody agent as described herein, a chimeric antigen receptor as described herein, or an immune effector cell , as described herein, a nucleic acid molecule as described herein, or a vector, as described herein), wherein the bispecific antibody consists of a first antigen-binding site based on a human antibody-based agent, as described herein, and the second antigen-binding site, which binds the immune cell, is contacted under conditions and for a time sufficient for tumor cell growth inhibition to occur (or for the immune cells to which the bispecific antibody binds to mediate tumor cell killing).

In some embodiments, the present invention provides a method for diagnosing a medical condition characterized by CD19 expression in a subject, the method comprising the step of administering an antibody agent (or a bispecific antibody or chimeric antigen receptor as described herein) as described herein and measuring binding said antibody agent (or bispecific antibody or chimeric antigen receptor) with a CD19 expressing cell in said subject.

In some embodiments of the method for diagnosing a medical condition, the method further includes the step of measuring one or more activities of one or more cells of said subject. In certain specific embodiments, one or more of the activities involve cell growth and/or apoptosis.

In some embodiments, the present invention provides a bispecific antibody consisting of a first antigen-binding site based on a human antibody agent as described herein and a second antigen-binding site that binds an immune cell for use in tumor growth inhibition.

In some embodiments, the present invention provides a bispecific antibody consisting of a first antigen-binding site based on a human antibody agent as described herein and a second antigen-binding site that binds an immune cell for use in killing tumor cells.

In some embodiments, the immune cell is a T cell or NK cell.

In some embodiments, the first and second antigen binding sites are scFv.

In some embodiments, the second antigen binding site binds CD3 on T cells.

In some embodiments, the present invention relates to a method of targeting T cells to kill CD19 expressing target cells, the method comprising the step of contacting one or more CD19 expressing target cells with one or more T cells and/or a bispecific antibody (or a human antibody-based agent as described herein, a chimeric antigen receptor as described herein, or an immune effector cell as described herein), wherein the bispecific antibody comprises the first antigen-binding site of the human antibody-based agent, as described herein, and a second antigen-binding site that binds CD3 on T cells, wherein the contact is made under conditions and for a time sufficient for the T cells to which the bispecific antibody binds to mediate the killing of target cells, expressing CD19. In certain specific embodiments, the first and second antigen binding sites are scFv.

In some embodiments, the present invention provides a bispecific antibody consisting of a first antigen-binding site based on a human antibody agent as described herein and a second antigen-binding site that binds CD3 on T cells, for use in targeting T cells to destruction of target cells expressing CD19.

In various embodiments, the linker sequence is or includes the sequence (G ₄ S) _n . In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

In various embodiments, the linker sequence is SRGGGGGGGGGGGGGGGGEMEMA (SEQ ID NO: 289).

In various embodiments, CD19 is human CD19.

In some embodiments, the present invention provides for the use of a human antibody, bispecific antibody, or chimeric antigen receptor (or immune effector cell expressing the chimeric antigen receptor) agent as described herein to treat or detect a condition associated with CD19 expression,

In some embodiments, the present invention provides for the use of a human antibody, bispecific antibody, or chimeric antigen receptor (or immune effector cell expressing the chimeric antigen receptor) as described herein in the manufacture of a medicament for use in medicine.

In some embodiments, the present invention provides for the use of a human antibody, bispecific antibody, or chimeric antigen receptor (or immune effector cell expressing the chimeric antigen receptor) as described herein in the manufacture of a medicament for use in a diagnostic test or assay.

In some embodiments, the present invention provides for the use of a human antibody, bispecific antibody, or chimeric antigen receptor (or immune effector cell expressing the chimeric antigen receptor) as described herein in the manufacture of a medicament for use in the diagnosis or treatment of cancer.

In some embodiments, the present invention provides for the use of an antibody based on a human antibody, a bispecific antibody, or a chimeric antigen receptor (or an immune effector cell expressing the chimeric antigen receptor) as described herein in the manufacture of a medicament for use in the diagnosis or treatment of a medical condition, characterized by the expression of CD19.

Brief description of the drawings

The drawings included in this specification, which consist of the following figures, are for illustrative purposes only and not for limitation.

In FIG. 1 shows exemplary phage clone binding to human CD19 for selected anti-CD19 agents based on a human antibody agent. In FIG. 1A shows exemplary binding of a phage clone (in OD ₄₅₀ ) of recombinant human CD19-Fc. In FIG. 1B shows exemplary phage clone binding (as mean fluorescence intensity, MFI) of human CD19 expressed on the cell surface. Control 1: non-CD19-binding scFv antibody phage clone; Control 2: commercial mouse anti-human CD19 antibody (Biolegend); Control 3: secondary antibody only.

In FIG. 2 shows typical mean fluorescence intensity (MFI) of phage clone binding to human CD19 positive Raji cells (Raji), CD19 knockout Raji cells (Raji-CD19 k.o.) and human CD19 negative Jurkat cells (Jurkat) in a flow-through assay. cytometry for selected anti-CD19 agents based on human antibodies. Control 1: secondary antibody only, including mouse anti-M13 antibody and PE-labeled anti-mouse antibody.

In FIG. 3 shows a representative SDS-PAGE image illustrating the selected bispecific antibodies described in Example 2. Lane numbers are indicated at the top of the gel image. Track 1: marker; Lane 2: CD19xCD3 bispecific antibody using clone 2; Lane 3: CD19xCD3 bispecific antibody using clone 3; Lane 4: CD19xCD3 bispecific antibody using clone 4; Lane 5: CD19xCD3 bispecific antibody using clone 37; Lane 6: BL19 (bispecific antibody for comparison, see SEQ ID NO: 30 in US Pat. No. 7,635,472).

In FIG. 4 shows exemplary octet association/dissociation curves for binding (y-axis, in nm) of exemplary bispecific antibody molecules to recombinant human CD19 ECD-Fc chimeric protein over time (x-axis, in seconds). Kinetic steps (separated by vertical dotted lines) are indicated above each sensory plot. a: base level; b: biotinylated human CD19-Fc (5 μg/ml); c: rebalancing; d: antibody binding to human CD19-Fc (10 μg/ml); e: antibody dissociation. The bifunctional antibodies tested included the CD19xCD3 bispecific antibody based on phage clones 4, 5, 6, 7, 37 and BL19 (comparative bispecific antibody, see SEQ ID NO: 30 in US Pat. No. 7,635,472).

In FIG. 5A shows a representative flow cytometric analysis of human PBMCs stained with CD20 (y-axis) and CD3 (x-axis) and the B cell population gate used for the bispecific antibody binding assay of FIG. 5V.

In FIG. 5B shows typical cancer cell kill (in % specific cell lysis, y-axis) of various cell lines for exemplary bispecific antibody molecules based on clones 4, 5, 6, and 37.

In FIG. 6A shows representative cell binding (measured by MFI in flow cytometry analysis) of exemplary antibody phage clone variants obtained by affinity maturation of individual parent antibody phage clones. Clone variants are indicated by a hyphen (i.e. clones 5-1, 5-3, 5-4, etc. were derived from the original clone 5, clones 6-1, 6-2, etc. were derived from the original clone 6). Asterisk: Clone 5-13 was not tested for binding to Jeko-1, aji-CD19 k.o. cell lines. or Jurkat.

In FIG. 6B shows representative cell binding of selected antibody phage clone variants compared to corresponding parental clones to Raji cells. Clone options are marked with a hyphen, i.e. clones 5-1, 5-3, 5-4, etc. were obtained from the original clone 5; clones 6-1, 6-2, etc. were derived from the original clone 6. The dotted lines indicate the original binding of the corresponding original clone with competitive binding of the corresponding bispecific antibody.

In FIG. 7A shows representative SDS-PAGE images illustrating selected bispecific antibodies constructed from affinity mature antibody phage clones as described in Example 4. Clone numbers are indicated in each lane.

In FIG. 7B shows representative cancer cell kill (in % specific cell lysis, y-axis) of various cell lines for selected bispecific antibody molecules constructed from human anti-CD19 affinity matured agents.

In FIG. 8 shows typical cancer cell kill (in % specific cell lysis, y-axis) of various cell lines for selected T cells expressing a chimeric antigen receptor (CAR-T) derived from human antibody-derived anti-CD19 agents. Target cell lines (CD19 ⁺ ) include Raji and Jeko-1; negative control cell lines (CD19 ^- ) include Jurkat and Raji-CD19 ko cells; BL19: CAR comparator based on SEQ ID NO: 30 of US Patent No. 7,635,472; FMC63: CAR comparator based on GenBank account number DD064902, US Pat. No. 7446179; Control: mock-transduced T cells.

In FIG. 9 shows typical CAR expression (upper panel) and in vitro cell killing (lower panel) of various cancer cell lines using selected anti-CD19 CAR-T or control T cells (sham-transduced). CD19 ⁺ cells: Raji, CA46, Jeko-1 and Daudi; CD19 ^- cells: Raji-CD19KO (i.e. Raji-CD19 ko), Jurkat, THP-1, HeLa, MDA-MB-231, MCF-7, SK-Hep-1 and HepG2.

In FIG. 10 shows representative cytokine release by mock-transduced T cells (mock-transduced) or T cells transduced with anti-CD19 CAR (Car based on clone 5-3) after co-culture with various CD19 ⁺ - (Raji, CA46) and CD19 ^- cell lines (Raji-K/O, ie Raji-CD19 ko, Jurkat).

Fig. 11 shows representative inhibition of tumor growth in vivo in NSG mice with exemplary anti-CD19 CAR-T clones 5-9 of a human anti-CD19 agent. Left: Representative photon emission kinetics in mice bearing NSG tumors, plotted and analyzed by area under the curve (AUC). Right: Representative photon emission images of mice bearing Raji-luc-GFP tumors at the third week of each treatment group.

In FIG. 12 shows FACS histograms of CD19 expression in 3T3 (left) and Raji (right) cell lines.

In FIG. 13 shows representative tumor bioluminescence from Raji lymphoma xenografts in NSG mice injected with an exemplary anti-CD19 CAR-T based on human anti-CD19 agent clones 5-13. Fig. 13A: Representative rainbow images of tumor bioluminescence from Raji lymphoma xenografts in NSG mice treated once with phosphate buffered saline (vehicle, n=5), T cells transduced without a CAR-coding construct (sham transduction, n=6 ) or T cells transduced with clones 5-13 of the anti-CD19-CAR-T coding construct (CAR-T 5-13, n = 7; 5x10 ⁶ CAR ⁺ T cells per mouse, days after tumor implantation , the dose was administered on day 5). The grayscale converted thermal plot shows the total photons/sec at the tumor site, which appear as dark spots superimposed on the mouse images; fig. 13B: Representative tumor growth quantification for sham-transduced and CAR-T 5-13 treatment groups presented as total flux (p/s) versus days after implantation of Raji lymphoma xenografts in NSG mice; C: Representative tumor growth quantification for sham-transduced and CAR-T 5-13 treatment groups presented as total flux (p/s) versus days post-reinfection in NSG mice re-infected with tumor cells seven weeks after initial implantation and treatment (n=3, 0.5×10 ⁶ Raji cells per mouse administered 35 days after initial implantation). Naive mice (n=2) were implanted with Raji cells one day after injection of 1x10 ⁶ mock-transduced T cells as a control.

In FIG. 14 shows representative tumor bioluminescence from NALM-6 leukemia xenografts in NSG mice injected with an exemplary anti-CD19 CAR-T based anti-CD19 agent human antibody, clones 5-13. Fig. 14A: Representative rainbow images of tumor bioluminescence from NALM-6 leukemia xenografts in NSG mice treated once with phosphate buffered saline (vehicle, n=6), T cells transduced without a CAR-coding construct (sham transduction, n=6 ), or T cells transduced with clones 5-13 of the anti-CD19 CAR-T coding construct (CAR-T 5-13, n = 6; 5×10 ⁶ CAR ⁺ T cells per mouse, days after tumor implantation; performed on day 5, days after tumor implantation, dosing was performed on day 5). Fig. 14B: Representative tumor growth quantification for vehicle, mock-transduced cells and CAR-T 5-13 treatment groups presented as total flux (p/s) versus days after implantation of NALM-6 leukemia xenografts in NSG mice.

In FIG. 15 shows typical CAR expression (lower panel) and in vitro cell killing (upper panel) of various cancer cell lines using selected anti-CD19 CAR-T or control T cells (sham-transduced). CD19 ⁺ cells: Raji, IM9 and Jeko-1; CD19 cells ^- : Raji-CD19KO (i.e. Raji-CD19 ko) and THP-1.

Definitions

The scope of the present invention is defined by the appended claims and is not limited to the specific embodiments described herein; those skilled in the art, upon reading the present disclosure, will be aware of various modifications that may be equivalent to such described embodiments or otherwise within the scope of the claims.

In General, the terminology used in this document corresponds to the understandable meaning in the art, unless expressly indicated otherwise. Explicit definitions of specific terms are given below; the meanings of these and other terms in specific cases within the scope of this description will be clear to those skilled in the art from the context.

In order to make the present invention more understandable, some terms are first defined below. Additional definitions for the following terms and other terms are provided in the description.

Administration: As used herein, the term "administration" refers to the administration of a composition to an object or system (eg, a cell, organ, tissue, organism, or a relevant part or set of parts). Those skilled in the art will appreciate that the route of administration may vary depending on, for example, the site or system into which the composition is administered, the nature of the composition, the purpose of administration, and so on. For example, in some embodiments, administration to an animal (e.g., human) subject may be bronchial (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intrahepatic, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intrauterine , intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and / or vitreal. In some embodiments, administration may include intermittent dosing. In some embodiments, the introduction may include continuous dosing (eg, perfusion) for at least a selected period of time.

Affinity. As is known in the art, "affinity" is a measure of the degree of interaction density with a particular ligand that binds to its partner. Affinities can be measured in different ways. In some embodiments, affinity is measured by quantitative analysis. In some such embodiments, the concentration of the binding partner may be fixed to exceed the concentration of the ligand to mimic physiological conditions. Alternatively or additionally, in some embodiments, the concentration of the binding partner and/or the concentration of the ligand may vary. In some such embodiments, affinity can be compared to a reference under comparable conditions (eg, concentrations).

Affinity matured (or affinity matured antibody): As used herein, the term refers to an antibody with one or more changes in one or more CDRs (or, in some embodiments, framework regions) that result in an improvement in the affinity of the antibody for an antigen, by compared with the original antibody, which does not have these changes. In some embodiments, affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies can be made by any of a variety of methods known in the art. Marks et al., 1992, BioTechnology 10: 779-783 describes affinity maturation by V _H and V _L domain shuffling. Random mutagenesis of CDRs and/or framework residues is described in: Barbas et al., 1994, Proc. Nat. Acad. Sci. USA 91:3809-3813; Schier et al., 1995, Gene 169: 147-155; Yelton et al., 1995. J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. Mol. Biol. 226:889-896. The choice of binders with improved binding properties is described by Thie et al., 2009, Methods Mol. Bio. 525:309-22.

Agent: As used herein, may refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. In some embodiments, the agent is or includes a natural product, as it was found and/or derived from nature. In some embodiments, the agent is or includes one or more objects that are human-made as they are designed, constructed and/or created through the action of human hands and/or are not found in nature. In some embodiments, the agent may be used in isolated or pure form; in some embodiments, the agent may be used in crude form. In some embodiments, potential agents are provided as collections or libraries, for example, which can be screened to identify or characterize active agents among them. Some specific embodiments of agents that may be used in accordance with the present invention include small molecule compounds, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA/RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptide mimetics etc. In some embodiments, the agent is or includes a polymer. In some embodiments, the agent is not a polymer and/or is substantially free of any polymer. In some embodiments, the agent includes at least one polymeric fragment. In some embodiments, the agent is absent or substantially free of any polymeric moiety.

Relief: As used herein, refers to the prevention, reduction, or mitigation of a condition, or the improvement of an object's condition. Relief includes, but does not require, complete recovery or complete prevention of the disease, disorder, or condition (eg, radiation injury).

Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and/or substance that can be included in a polypeptide chain. In some embodiments, the amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in natural peptides. "Non-standard amino acid" refers to any amino acid other than standard amino acids, whether synthetically derived or derived from a natural source. As used herein, the term "synthetic amino acid" encompasses chemically modified amino acids, including, without limitation, salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can alter the half-life of the peptide without adversely affecting their activity. Amino acids can participate in a disulfide bond. Amino acids may contain one or post-translational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, fragments of biotin, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and/or an amino acid residue of a peptide. It will be clear from the context in which this term is used whether it refers to a free amino acid or a peptide residue.

Animal: As used herein, refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, of any gender, and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In some embodiments, the non-human animal is a mammal (eg, a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate, and/or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, the animal may be a transgenic animal, a genetically modified animal, and/or a clone.

Antibody: As used herein, the term has the meaning of the art and refers to an immunoglobulin (Ig) that specifically binds to a particular antigen. As known to those skilled in the art, naturally produced antibodies typically consist of four polypeptide chains, two heavy (H) chains and two light (L) chains. Each heavy and light chain consists of a variable region (abbreviated as HCVR or V _H and LCVR or V _L , respectively) and a constant region. The heavy chain constant region includes the C _H 1, C _H 2 and C _H 3 domain (and optionally the C _H 4 domain for IgM and IgE). The light chain constant region consists of a single domain, C _L . In V _H and V _L , the region further includes regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (eg, IgM, IgD, IgG, IgA, and IgE), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a specific antigen. In some embodiments, the term encompasses any polypeptide with sufficient immunoglobulin building blocks to confer specific binding. In various embodiments, suitable antibody agents may include, but are not limited to, monoclonal antibodies, polyclonal antibodies, humanized antibodies, primatized antibodies, chimeric antibodies, human antibodies, bispecific or multispecific antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or their fragments), conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radioactive labels, cytotoxins), small modular immunopharmaceuticals (SMIPsTM), single chain antibodies, camel antibodies, antibody fragments, etc. In some embodiments, this term may refer to a cross-linked peptide. In some embodiments, this term may refer to an antibody-like peptidomimetic. In some embodiments, this term may refer to an antibody-like binding scaffold protein. In some embodiments, this term may refer to monobodies or adnectins. In many embodiments, the antibody agent is a polypeptide or a polypeptide thereof, the amino acid sequence of which includes one or more structural elements recognized by those skilled in the art as a complementarity determination region (CDR); in some embodiments, the antibody agent is or includes a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is essentially identical to that found in the control antibody (eg, parent antibody). In some embodiments, the included CDR is substantially identical to the reference CDR in that it is either identical in sequence or has 1 to 5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid within the included CDR is removed, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. . In some embodiments, the included CDR is substantially identical to the reference CDR in that 1-5 amino acids in the included CDR are removed, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid within the included CDR is changed from the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1-5 amino acids within the included CDR are substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is or includes a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is a polypeptide comprising all of the CDRs found in a particular reference antibody chain or chains (eg, heavy chain and/or light chain).

antibody component. As used herein, the term refers to a polypeptide element (which may be a complete polypeptide or part of a larger polypeptide such as, for example, a chimeric polypeptide as described herein) that specifically binds to an epitope or antigen and includes one or more structural signs of immunoglobulin. In general, an antibody component is any polypeptide whose amino acid sequence includes elements characteristic of an antibody binding region (e.g., an antibody light chain or its variable region or one or more complementarity determining regions ("CDRs") or heavy chain of an antibody or its variable region). or one or more CDRs, optionally in the presence of one or more framework regions). In some embodiments, the antibody component is or includes a full length antibody. In some embodiments, the antibody component is less than complete, but includes at least one binding site (comprising at least one, and preferably at least two, known antibody "variable region" sequences). In some embodiments, the term "antibody component" encompasses any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. In specific embodiments, the included "antibody component" encompasses polypeptides having a binding domain that shows at least 99% identity with an immunoglobulin binding domain. In some embodiments, the included "antibody component" is any polypeptide having a binding domain that shows at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity with an immunoglobulin binding domain. , for example, with a reference immunoglobulin binding domain. The included "antibody component" may have an amino acid sequence identical to the amino acid sequence of an antibody (or portion thereof, such as its antigen-binding portion) that is found in a natural source. The antibody component may be monospecific, bispecific, or multispecific. The antibody component may include structural elements characteristic of any class of immunoglobulin, including any of the human classes: IgG, IgM, IgA, IgD and IgE. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Such antibody embodiments may also be in bispecific, dual specific, or multispecific formats; specifically binding to two or more different antigens. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of V _H , V _L , C _H 1 and C _L domains; (ii) an F(ab') ₂ fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of V _H and C _H 1 domains; (iv) an Fv fragment consisting of the V _H and V _L domains of a single antibody arm, (v) a dAb fragment (Ward et al, 1989, Nature 341: 544-546) that includes one variable domain; and (vi) a dedicated complementarity determination region (CDR). In addition, although the two domains of the Fv fragment V _H and V _L are encoded by separate genes, they can be combined, using recombinant methods, with a synthetic linker that allows them to be converted into a single protein chain in which the V _H and V _L pairs form monovalent molecules ( known as single chain Fvs (scFvs), see for example Bird et al., 1988, Science 242: 423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883). In some embodiments, an "antibody component" as described herein is or includes such a single chain antibody. In some embodiments, the "antibody component" is or includes a diabody. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on the same polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, thereby causing the domains to pair with complementary domains of the other strand and create two antigen-binding sites (see, for example, Holliger, P., et al. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al., 1994, Structure 2:1121-1123 ). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering, 2001, Springer-Verlag. New York. 790 pp. ISBN 3-540-41354-5). In some embodiments, the antibody component is or includes a single chain "linear antibody" containing a pair of tandem Fv segments (V _H -C _H 1 -V _H -C _H 1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., 1995, Protein Eng., 8 (10): 1057-1062 and US Pat. No. 5,641,870). In some embodiments, the antibody component may have structural elements characteristic of chimeric, humanized, or human antibodies. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determination region (CDR) of the recipient are replaced with CDR residues of a non-human (donor) antibody, such as mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some embodiments, the antibody component may have structural elements characteristic of a human antibody. In some embodiments, an antibody component may be included in the chimeric molecule, wherein the chimeric molecule further includes one or more additional polypeptides or polypeptide fragments (eg, a signal component, a transmembrane component, etc.).

Antibody fragment: As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; triatela; tetrabodies; linear antibodies; single chain antibody molecules; and CDR-containing groups included in multispecific antibodies formed from antibody fragments. Those of skill in the art will appreciate that the term "antibody fragment" is not intended to imply or be limited to any particular method of preparation. An antibody fragment can be generated using any suitable methodology, including, but not limited to, intact antibody digestion, chemical synthesis, recombinant production, and the like.

An antibody polypeptide. As used herein, the term "antibody polypeptide" or "antibody" or antigen-binding fragment thereof, which may be used interchangeably, refers to a polypeptide(s) capable of binding to an epitope. In some embodiments, the antibody polypeptide is a full length antibody, and in some embodiments it is less than full length but includes at least one binding site (comprising at least one and preferably at least two sequences with the structure "variable regions" of an antibody). In some embodiments, the term "antibody polypeptide" encompasses any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. In specific embodiments, "antibody polypeptides" include polypeptides having a binding domain that shows at least 99% identity with an immunoglobulin binding domain. In some embodiments, an "antibody polypeptide" is any protein having a binding domain that shows at least 70%, 80%, 85%, 90%, or 95% identity with an immunoglobulin binding domain, e.g., a reference immunoglobulin binding domain. . The included "antibody polypeptide" may have an amino acid sequence identical to the amino acid sequence of an antibody that is found in a natural source. Antibody polypeptides of the present invention may be prepared by any available means, including, for example, isolation from a natural source or antibody library, recombinant production in or with a host system, chemical synthesis, and the like. or their combinations. An antibody polypeptide may be monoclonal or polyclonal. An antibody polypeptide may be a member of any class of immunoglobulins, including any of the human IgG, IgM, IgA, IgD, and IgE classes. In some embodiments, the antibody may be a member of the IgG immunoglobulin class. As used herein, the terms "antibody polypeptide" or "antibody characteristic" are used interchangeably and refer to any antibody derivative that has the ability to bind to an epitope of interest. In some embodiments, an "antibody polypeptide" is an antibody fragment that retains at least a significant portion of the specific binding capacity of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , scFv, Fv, dsFv diabody, and Fd fragments. Alternatively or additionally, an antibody fragment may contain multiple chains that are linked, for example, by disulfide bonds. In some embodiments, the antibody polypeptide may be a human antibody. In some embodiments, the antibody polypeptide may be a humanized antibody. Humanized antibody polypeptides include chimeric immunoglobulins, immunoglobulin chains, or antibody polypeptides (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding antibody subsequences) that contain a minimal sequence derived from a non-human immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced with CDR residues from a non-human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity and capacity.

Antigen binding fragment. The term "antigen-binding fragment", as used herein, refers to one or more antibody fragments that retain the ability to bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of V _L , V _H , C _L and C _H 1 domains; (ii) an F(ab') ₂ fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of V _H and C _H 1 domains; (iv) an Fv fragment consisting of the V _L and V _H domains of one antibody arm, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) which consists of the V _H domain; (vi) a dedicated complementarity determination region (CDR), such as a V _H CDR3, with or without an additional sequence (linker, framework(s), etc.) and (vii) a combination of two to six dedicated CDRs containing or not containing an additional sequence (linker, frame region, etc.). In addition, although the two regions of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined, using recombinant methods, with a synthetic linker that allows a single protein chain in which the VL and VH pairs form monovalent molecules (known as single-stranded Fv (scFv), see, for example, Bird et al (1988) Science 242:423-426 and Huston et al (1988) Proc Natl Acad Sci USA 85:5879-5883). Such single chain antibodies are also intended to be included in the term "antigen binding fragment" of an antibody. In addition, antigen binding fragments include chimeric immunoglobulin binding domain proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) that is fused with an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain C _H 2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain C _H 3 constant region fused to _CH 2 . or a few cysteine residues with serine residues to prevent dimerization. Such chimeric immunoglobulin binding domain proteins are further disclosed in US Patent Application Publication No. 2003/0118592 A1 and US Patent Application Publication No. 2003/0133939 A1. These antibody fragments are prepared by conventional methods known to those skilled in the art and the fragments are screened for utility in the same manner as intact antibodies. In addition, antigen binding fragments include divalent (or divalent) single chain variable fragments (di-scFv, bi-scFv) or otherwise so-called diabodies, which can be constructed by standard molecular biological tools.

Biological activity: As used herein, the term refers to an observable biological effect or result achieved by an agent or object of interest. For example, in some embodiments, specific binding is a biological activity. In some embodiments, modulation (eg, induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is assessed by detecting a direct or indirect biologically produced product or event of interest.

Bispecific antibody: As used herein, the term refers to a bispecific binding agent in which at least one, and typically both, of the binding moiety is or contains an antibody component. Many different bispecific antibody structures are known in the art. In some embodiments, each binding fragment in a bispecific antibody that is or includes an antibody component comprising V _H and/or V _L regions; in some such embodiments, the V _H and/or V _L regions are regions found in a particular monoclonal antibody. In some embodiments, when the bispecific antibody comprises two antibody binding groups, each includes V _H and/or V _L regions from different monoclonal antibodies.

Bispecific binding agent: As used herein, the term refers to a polypeptide agent with two discrete binding groups, each binding to a different target. In some embodiments, the bispecific binding agent is a single polypeptide; in some embodiments, the bispecific binding agent is or includes a plurality of peptides, which in some such embodiments may be covalently linked to each other, such as by crosslinking. In some embodiments, the two binding groups of the bispecific binding agent recognize different sites (eg, epitopes) on the same target (eg, antigen); in some embodiments, they recognize different targets. In some embodiments, the bispecific binding agent is capable of simultaneously binding to two targets that have different structures.

Carrier: As used herein, the term refers to the diluent, adjuvant, excipient, or vehicle with which the composition is administered. In some exemplary embodiments, carriers may include sterile liquids such as, for example, water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some embodiments, the carriers are or include one or more solid components.

CDR: As used herein, the term refers to the complementarity-determining region in the variable region of an antibody. There are three CDRs in each of the heavy chain and light chain variable regions, which are designated as CDR1, CDR2 and CDR3 for each of the variable regions. A "set of CDRs" or "CDR set" refers to a group of three or six CDRs that occur either in a single variable region capable of binding to an antigen, or CDRs of cognate heavy and light chain variable regions capable of binding an antigen. CDR boundaries have been defined differently depending on the system, of which several are known in the art (eg Kabat, Chothia, IMGT, etc.).

Chimeric Antigen Receptors (CAR): The antibody agents of the present invention, including single chain variable fragments (scFv), can be used to produce chimeric antigen receptors, the preparation and use of which is well known in the art. A chimeric antigen receptor (CAR) is typically an artificially engineered fusion protein or polypeptide containing the antigen-binding domain of an scFv or other antibody agent coupled to the signaling domains of an immune cell (eg, T cells or NK cells). Characteristics of CARs include their ability to redirect the specificity and reactivity of an immune cell (e.g., T cells or NK cells) to a chosen target, either in MHC-restricted (in the case of TCR-mimic antibodies) or MHC-unrestricted (in the case of anti- cell surface proteins) using the antigen-binding properties of monoclonal antibodies. MHC-unrestricted antigen recognition gives CAR-expressing immune cells (eg, T cells or NK cells) the ability to recognize antigen independent of antigen processing, thereby bypassing the primary mechanism for tumor escape from killing.

Chimeric antigen receptor (CAR) therapy (or adoptive cell therapy): As used herein, the term refers to the use of chimeric antigen receptors for therapeutic treatment, including, for example, adoptive cell therapy. Adoptive cell therapy is a therapeutic approach that typically involves isolating and ex vivo expanding and/or manipulating immune cells (eg NK cells or T cells) and then administering these cells to a patient, eg for cancer treatment. The introduced cells can be autologous or allogeneic. Cells can be manipulated to express chimeric antigen receptors by any of the known methods, including, for example, the use of RNA and DNA transfection, viral transduction, electroporation, all of which are technologies known in the art.

Comparable: As used herein, refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to each other, but that are similar enough to allow a comparison between them to be reasonable. draw conclusions based on differences or similarities. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of distinct features. Those skilled in the art will understand in context what degree of identity is required in any case for two or more such agents, entities, situations, condition sets, etc. that are considered comparable. For example, those skilled in the art will recognize that sets of circumstances, individuals, or populations are comparable to each other when they have a sufficient number and type of substantially identical features to provide a reasonable inference that differences in outcomes or phenomena observed under or with different sets of circumstances, individuals or populations are caused by or indicate a change in various characteristics.

Control. As used herein, the term refers to the art-understood meaning of "control", which is the standard against which results are compared. Typically, controls are used to enhance the integrity of experiments by highlighting variables in order to infer about those variables. In some embodiments, the control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator. As used herein, the term "control" may refer to "control antibody". A "control antibody" can be a human, chimeric, humanized CDR-grafted, multispecific, or bispecific antibody as described herein, an antibody that differs, as described herein, in an antibody fragment or antibody component, or in the parent antibody. In one experiment, a "test" (i.e., the variable being tested) is applied. In the second experiment, the "control", the test variable, is not used. In some embodiments, the control is a historical control (ie, a test or assay performed previously, or an amount or result that was previously known). In some embodiments, the control is or includes a printed or other stored record. The control can be positive or negative.

Relevant: As used herein, refers to the position/identity of an amino acid residue in a polypeptide of interest. Those of ordinary skill in the art will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that the amino acid "corresponding" to the residue at position 190, for example, need not be the ^190th amino acid in a particular amino acid. chain, but rather corresponds to the residue found at 190 in the reference polypeptide; those skilled in the art readily understand how to identify the "corresponding" amino acids.

Object/detecting agent: As used herein, the term refers to any element, molecule, functional group, compound, fragment or part that can be detected. In some embodiments, the discovery object is provided or used separately. In some embodiments, a discovery object is provided and/or shared (eg, connected to) with another agent. Examples of detecting objects include, without limitation: various ligands, radionuclides (e.g., ³ H, ¹⁴ C, ¹⁸ F, ¹⁹ F, ³² P, ³⁵ S, ¹³⁵ I, ¹²⁵ I, ¹²³ I, ⁶⁴ Cu, ¹⁸⁷ Re, ¹¹¹ In, ⁹⁰ Y, ⁹⁹ mTc, ¹⁷⁷ Lu, ⁸⁹ Zr, etc.), fluorescent dyes (for specific illustrative fluorescent dyes, see below), chemiluminescent agents (such as, for example, acridine esters, stabilized dioxanes, and (for example , gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes (see below for specific examples of enzymes), colorimetric labels (such as, for example, dyes, colloidal gold, etc.), biotin, dioxygenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

Effector function: As used herein, the term refers to a biochemical event that results from the interaction of the Fc region of an antibody with an Fc receptor or ligand. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-associated cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). In some embodiments, the effector function is a function that acts after antigen binding, that acts independently of antigen binding, or both.

Effector cell: As used herein, the term refers to a cell of the immune system that mediates one or more effector functions. In some embodiments, effector cells may include, but are not limited to, one or more monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, T lymphocytes, B-lymphocytes and can be from any organism, including, without limitation, humans, mice, rats, rabbits, and monkeys.

Constructed: As used herein, generally refers to the aspect of manipulating a human hand. For example, in some embodiments, a polynucleotide may be considered "engineered" when two or more sequences that are not naturally related to each other in that order are manipulated by human hands so as to be directly related to each other in the engineered polynucleotide. In some specific such embodiments, the engineered polynucleotide may contain a regulatory sequence that is naturally operably linked to the first coding sequence, but not operably linked to a second coding sequence that is hand-linked such that it is operably linked to the second coding sequence. Alternatively or additionally, in some embodiments, the first and second nucleotide sequences, each of which encodes polypeptide elements or domains that are not naturally associated with each other, may be associated with each other in a single engineered polynucleotide. Likewise, in some embodiments, a cell or organism may be considered "engineered" if it has been processed so that its genetic information has been altered (for example, new genetic material has been introduced that was not previously present, or previously present genetic material has been altered or removed. ). According to common practice and as understood by those skilled in the art, the progeny of an engineered polynucleotide or cell is generally still referred to as "engineered" even if the actual manipulation was performed on the previous entity. In addition, as will be appreciated by those skilled in the art, various methodologies are available by which the "design" described herein can be achieved. For example, in some embodiments, "design" may include selecting or designing (e.g., nucleic acid sequences, polypeptide sequences, cells, tissues, and/or organisms) using computer systems programmed to analyze or compare, or in other words to analyze, recommend and/or selection of sequences, changes, etc.). Alternatively or additionally, in some embodiments, "engineered" may include the use of in vitro chemical synthesis methodologies and/or recombinant nucleic acid technologies, such as, for example, nucleic acid amplification (e.g., via polymerase chain reaction), mutation, transformation, transfection, etc. .d. and/or any of a variety of controlled mating techniques. As will be understood by those skilled in the art, many known methods (e.g., for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.)) are well known in the art and described in various general and more specific sources that are cited and/or discussed in the present description. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Epitope: As used herein, the term includes any fragment that is specifically recognized by an immunoglobulin binding component (eg, antibody or receptor). In some embodiments, an epitope consists of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are exposed on the surface when the antigen assumes the appropriate three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of these chemical atoms are groups that are physically separated from each other when the antigen assumes an alternative conformation (eg, linearizes).

Excipient: As used herein, the term refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or promote a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene, glycol, water, ethanol and the like.

Fc ligand. As used herein, the term refers to a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc ligand complex. Fc ligands include, but are not limited to, FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), FcγRIIIB (CD16B), FcγRI (CD64), FcεRII (CD23), FcRn, Clq, C3, staphylococcal protein A, streptococcal protein G and viral FcγR. Fc ligands may include as yet undiscovered molecules that bind to Fc.

Framework or framework region: as used herein, refers to the sequences of the variable region minus the CDR. Since the CDR sequence may be defined by different systems, similarly, the framework sequence is subject to different interpretations as appropriate. Six CDRs divide the framework regions into heavy and light chains in four sub-regions (FR1, FR2, FR3 and FR4) on each chain in which CDR1 is between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying sub-regions such as FR1, FR2, FR3 or FR4, a framework region, as mentioned by others, represents the combined FRs within the variable region of a single natural immunoglobulin chain. As used herein, FR is one of four subregions, for example FR1 is the first framework region closest to the amino terminus of the variable region and 5' to CDR1, and FRs are two or more subregions that make up the framework.

Host cell: As used herein, the term refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those of skill in reading this disclosure will appreciate that such terms refer not only to the particular cell being tested, but also to the progeny of such a cell. Because certain changes may occur in subsequent generations due to mutations or environmental influences, such progeny may not be essentially identical to the original cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, the host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms that are suitable for the expression of exogenous DNA (eg, a recombinant nucleic acid sequence). Typical cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g. strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g. S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells or chimeric cells such as, for example, hybridomas or quadromas. In some embodiments, the host cell is a human, primate, higher primate, hamster, rat, or mouse cell. In some embodiments, the host cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO Kl, DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney ( e.g. HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g. BHK21), Jurkat, Daudi, A431 (epidermal), CV -1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, Sertoli cells, BRL 3 cells, HT1080 cells, myeloma cells, tumor cells and a cell line derived from the above cell. In some embodiments, the host cell includes one or more viral genes, such as a retinal cell that expresses a viral gene (eg, a PER.C6™ cell).

Human antibody: As used herein, the term is intended to include antibodies having variable and constant regions generated (or assembled) from human immunoglobulin sequences. In some embodiments, antibodies (or antibody components) may be considered "human" even if their amino acid sequences include residues or elements not encoded by germline human immunoglobulin sequences (e.g., include sequence variations, e.g., which may (initially) have been introduced by chance or site-directed mutagenesis in vitro or somatic mutation in vivo), for example, in one or more CDRs and in particular CDR3.

Humanized: As is known in the art, the term "humanized" is commonly used to refer to antibodies (or antibody components) whose amino acid sequence includes the V _H and V _L region sequences from a reference antibody grown in a non-human species (e.g. mouse) , but also includes modifications to these sequences relative to a reference antibody designed to make them more "human-like", that is, more similar to human germline variable sequences. In some embodiments, a "humanized" antibody (or antibody component) is an antibody that immunospecifically binds to an antigen of interest and that has a framework (FR) region having a substantially amino acid sequence, such as the amino acid sequence of a human antibody, and a complementarity-determining region (CDR) having a substantially amino acid sequence such as that of a non-human antibody. A humanized antibody comprises essentially all of at least one and typically two variable domains (Fab, Fab', F(ab') ₂ , FabC, Fv) in which all or substantially all of the CDRs correspond to immunoglobulin regions, non-human (i.e., donor immunoglobulin) and all or substantially all of the framework regions are human immunoglobulin consensus sequence. In some embodiments, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. In some embodiments, the humanized antibody includes both a light chain and at least a heavy chain variable domain. The antibody may also include _CH 1, hinge, _CH 2, _CH 3 and optionally the _CH 4 region of the heavy chain constant region. In some embodiments, a humanized antibody only includes a humanized V _L region. In some embodiments, a humanized antibody only includes a humanized V _H region. In some specific embodiments, a humanized antibody includes humanized V _H and V _L regions.

Hydrophilic. As used herein, the term "hydrophilic" and/or "polar" refers to the tendency to mix or readily dissolve in water.

Hydrophobic: As used herein, the term "hydrophobic" and/or "non-polar" refers to a tendency to repel, not combine with, or inability to readily dissolve in water.

Improve, Increase, or Reduce: When used herein, or their grammatical equivalents, indicate values that refer to a baseline measurement, such as a measurement in the same individual prior to treatment described herein, or a measurement in a control individual (or several control individuals) in the absence of the treatment described herein. A "control individual" is an individual suffering from the same form of disease or injury as the individual being treated.

In vitro: As used herein, the term refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., and not in a multicellular organism.

In vivo: As used herein, the term refers to events that occur in a multicellular organism such as a human and a non-human animal. In the context of cellular systems, this term can be used to refer to events occurring in a living cell (as opposed to, for example, in vitro systems).

Isolated: As used herein, the term refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was originally produced (whether in nature and/or experimentally installation), and/or (2) with which they were designed, produced, obtained and/or made by human hands. The isolated substances and/or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91% about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than 99% of the other components they were originally associated with. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after it has been combined with certain other components, such as, for example, one or more carriers or excipients ( eg buffer, solvent, water, etc.); in such embodiments, the percentage recovery or purity of a substance is calculated without including such carriers or excipients. To give just one example, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, is considered "isolated" when: a) by virtue of its origin or source of origin, it is not associated with some or all of the components that accompany it in a natural state by nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that naturally produce it; c) is expressed or otherwise associated with components from a cell or other expression system that is not of the species that naturally produces them. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than that which naturally produces it is considered an "isolated" polypeptide. Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification methods may be considered an "isolated" polypeptide to the extent that it has been separated from the other components a) with which it is naturally associated; and/or b) with which it was associated during initial production.

K _D : As used herein, the term refers to the dissociation constant of a binding agent (eg, an antibody agent or its binding component) from a complex with its partner (eg, an epitope to which the antibody agent or its binding component binds).

k _off : As used herein, the term refers to the dissociation rate constant of a binding agent (eg, an antibody agent or binding component thereof) from a complex with its partner (eg, an epitope to which the antibody agent or binding component thereof binds).

k _on : As used herein, the term refers to the rate constant of association of a binding agent (eg, an antibody agent or its binding component) with its partner (eg, an epitope to which the antibody agent or its binding component binds).

Linker: As used herein, the term is used to refer to that part of a multi-element polypeptide that connects different elements to each other. For example, those skilled in the art believe that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them together. In some embodiments, the polypeptide containing the linker element has the general structure of the general form S1-L-S2, where S1 and S2 may be the same or different and represent two domains linked to each other by a linker. In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized in that it does not assume a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. Many different linker elements that can be appropriately used in the design of polypeptides (eg, chimeric polypeptides) are known in the art (see, for example, Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak, R. J. et al., 1994, Structure 2:1121-1123).

Multivalent binding agent (or multispecific binding agent): As used herein, the term refers to a binding agent capable of binding to two or more antigens, which may be on the same molecule or on different molecules. The multivalent binding agents described herein are, in some embodiments, designed to have two or more antigen-binding sites and are generally not naturally occurring proteins. Multivalent binding agents described herein refer to binding agents capable of binding two or more related or unrelated targets. Multivalent binding agents may consist of multiple copies of a single antibody component or multiple copies of different antibody components. Such binding agents are capable of binding to two or more antigens and are tetravalent or multivalent binding agents. Multivalent binding agents may additionally contain a therapeutic agent such as, for example, an immunomodulator, a toxin, or an RNase. Multivalent binding agents as described herein, in some embodiments, are capable of simultaneously binding to at least two targets that have different structures, such as two different antigens, two different epitopes on the same antigen, or a hapten and/or antigen or epitope. In many embodiments, the multivalent binding agents of the present invention are proteins designed with the characteristics of the multivalent binding agents described herein. The multivalent binding agents of the present invention may be monospecific (able to bind one antigen) or multispecific (able to bind two or more antigens) and may consist of two heavy chain polypeptides and two light chain polypeptides. Each binding site, in some embodiments, consists of a heavy chain variable domain and a light chain variable domain with a total of six CDRs involved in antigen binding per antigen binding site.

Nucleic acid. As used herein, in its broadest sense, refers to any compound and/or substance that is or can be included in an oligonucleotide chain. In some embodiments, the nucleic acid is a compound and/or substance that is or can be incorporated into the oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (eg, nucleotides and/or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide strand containing individual nucleic acid residues. In some embodiments, "nucleic acid" is or includes RNA; in some embodiments, "nucleic acid" is or includes DNA. In some embodiments, the nucleic acid is, includes, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, the nucleic acid is, includes, or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid analog differs from the nucleic acid in that it does not use a phosphodiester backbone. For example, in some embodiments, the nucleic acid is or consists of one or more "peptide nucleic acids" that are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone are contemplated within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is or consists of one or more naturally occurring nucleosides (eg, adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is composed of one or more nucleoside analogs (examples are 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl- uridine, 2-aminoadenosine, C5-bromuridine, C5-fluorouridine, C5-ioduridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid includes one or more modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those found in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or protein. In some embodiments, the nucleic acid includes one or more introns. In some embodiments, nucleic acids are produced by one or more isolations from a natural source, enzymatic synthesis by complementary template polymerization (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid has at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more lengths. In some embodiments, the nucleic acid is single stranded; in some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence containing at least one element that encodes or is complementary to a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

Functionally related: As used herein, refers to an association in which the described components are in a relationship that allows them to function in the intended manner. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences that are adjacent to the gene of interest and expression control sequences that act in trans or at a distance to control said gene of interest. As used herein, the term "expression control sequence" refers to polynucleotide sequences that are necessary to effect expression and processing of the coding sequences to which they are ligated. Expression control sequences include the appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase the efficiency of translation (ie the Kozak consensus sequence); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences differs depending on the host organism. For example, in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence, while in eukaryotes, such control sequences typically include promoters and a transcription termination sequence. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences.

Peptide. The term "peptide" refers to two or more amino acids connected to each other by peptide bonds or modified peptide bonds. In specific embodiments, "peptide" refers to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 20 amino acids, or less than about 10 amino acids.

Physiological conditions: As used herein, the term has the meaning understood in the art, referring to the conditions under which cells or organisms live and/or reproduce. In some embodiments, the term refers to the conditions of the external or internal environment, which may occur in nature for an organism or cellular system. In some embodiments, physiological conditions are those conditions that are present in a human or non-human animal, especially those conditions that are present at and/or in the surgical site. Physiological conditions typically include, for example, temperature range 20-40° C., atmospheric pressure 1, pH 6-8, glucose concentration 1-20 mM, oxygen concentration at atmospheric levels, and gravity as it occurs on Earth. In some embodiments, laboratory conditions are controlled and/or maintained under physiological conditions. In some embodiments, physiological conditions occur in the body.

Polypeptide. As used herein, the term refers to any polymer chain of amino acids. In some embodiments, the polypeptide has an amino acid sequence that occurs naturally. In some embodiments, the polypeptide has an amino acid sequence that does not occur naturally. In some embodiments, the polypeptide has an amino acid sequence that is designed to be designed and/or produced by the action of human hands. In some embodiments, the polypeptide may contain or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, the polypeptide may contain or consist of only natural amino acids or only non-natural amino acids. In some embodiments, the polypeptide may include D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may contain only D-amino acids. In some embodiments, the polypeptide may contain only L-amino acids. In some embodiments, the polypeptide may include one or more pendant groups or other modifications, such as modification or addition to one or more amino acid side chains, at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, the polypeptide may be cyclic and/or may contain a cyclic moiety. In some embodiments, the polypeptide is non-cyclic and/or does not include any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or contain a cross-linked polypeptide. In some embodiments, the term "polypeptide" may be attached to the name of a reference polypeptide, activity, or structure; in such cases, it is used herein to refer to polypeptides that share a corresponding activity or structure and thus can be considered members of the same class or family of polypeptides. For each such class, exemplary polypeptides within the class are provided herein and/or those skilled in the art will know whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for a class of polypeptide. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a given range) with a reference class polypeptide; in some embodiments with all polypeptides within a class). For example, in some embodiments, the representative polypeptide shows an overall degree of homology or sequence identity with the reference polypeptide that is at least 30% to 40%, and often exceeds about 50%, 60%, 70%, 80%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or includes at least one area (i.e., a conservative area that can in some embodiments be or include a characteristic sequence element) that exhibits very high sequence identity, often in excess of 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically spans at least three to four and often up to 20 or more amino acids; in some embodiments, the conserved region spans at least one region of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a useful polypeptide may contain or consist of a fragment of the original polypeptide. In some embodiments, a useful polypeptide that may contain or consist of multiple fragments, each of which is located in the same parent polypeptide in a different spatial arrangement relative to each other than in the polypeptide of interest (for example, fragments that are directly linked in the parent peptide, may be spatially separated in the polypeptide of interest or vice versa, and/or the fragments may be present in a different order in the polypeptide of interest than in the parent), such that the polypeptide of interest is a derivative of its parent polypeptide.

Prevent or prevent: When used herein in connection with the occurrence of a disease, disorder and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or delaying the onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when the onset of a disease, disorder or condition has been delayed for a predetermined period of time.

Recombinant: as used herein, is intended to refer to polypeptides (e.g., antibodies or antibody components or multispecific binding agents as described herein) that are engineered, engineered, produced, expressed, generated, or isolated by recombinant means, e.g., as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a recombinant combinatorial human polypeptide library (Hoogenboom HR, 1997, TIB Tech. 15: 62-70; Azzazy H. and Highsmith WE, 2002, Clin. Biochem. 35:425-45; Gavilondo JV, and Larrick JW, 2002, BioTechniques 29:128-45; Hoogenboom H., and Chames P., 2000, Immunol. Today 21:371-8), antibodies isolated from an animal (e.g. , mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor, LD, et al., 1992, Nucl. Acids Res. 20:6287-95; Kellermann SA., and Green LL, 2002, Curr. Opin. Biotech. 13:593-7; Little, M. et al., 2000, Immunol. Today 21:364-70; Murphy, AJ et al., 2014, Proc. Natl. Acad. sci. USA 111 (14): 5153-8) or polypeptides obtained, expressed, created or isolated by any other method, which includes splicing selected sequence elements with each other. In some embodiments, one or more of these selected sequence elements occur naturally. In some embodiments, one or more of these selected sequence elements are designed in silico. In some embodiments, one or more of these selected sequence elements are the result of mutagenesis (eg, in vivo or in vitro) of a known sequence element, eg, from a natural or synthetic source. For example, in some embodiments, the recombinant antibody polypeptide consists of sequences found in the germline of the source organism of interest (eg, human, mouse, etc.). In some embodiments, the recombinant antibody has an amino acid sequence that is the result of mutagenesis (e.g., in vitro or in vivo, e.g., in a transgenic animal) such that the amino acid sequences of the V _H and V _L regions of the recombinant antibody are sequences that, being the source and associated with germline V _H and V _L sequences may not naturally exist in the in vivo germline antibody repertoire.

Reference: When used in this document, describes a standard, control, or other appropriate reference against which a comparison is made, as described in this document. For example, in some embodiments, a reference agent, animal, subject, population, sample, sequence, set of steps, set of conditions, or value against which the agent, animal, subject, population, sample, sequence, series of steps of interest is compared , condition set, or value. In some embodiments, the benchmark is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, the reference is a historical reference, optionally embodied in a material environment. Typically, as will be understood by those skilled in the art, a benchmark is defined or characterized under conditions comparable to those used in the assessment of interest.

specific binding. As used herein, the term refers to the ability of a binding agent to discriminate between possible partners in the environment in which the binding is to take place. It is believed that a binding agent that interacts with one specific target, in the presence of other potential targets, "specifically binds" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the partner binding agent complex; in some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by conducting such detections or determinations over a range of concentrations. In some embodiments, specific binding is assessed by determining the difference in binding affinity between cognate and non-cognate targets. For example, the binding agent may have a binding affinity for a cognate target that is about 3, 4, 5, 6, 7, 8, 9, 10 or more than a binding affinity for a non-cognate target.

Specificity. As known in the art, "specificity" is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.

Object: As used herein, the term means any mammal, including humans. In some embodiments of the present invention, the subject is an adult, adolescent, or infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "object". Also according to the present invention, the administration of pharmaceutical compositions and/or the effectiveness of methods of treatment in utero is provided.

Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting a general or nearly complete measure or degree of a characteristic or property of interest. Any ordinary biologist will understand that biological and chemical phenomena rarely, if ever, come to an end and/or come to an end or reach or avoid an absolute result. Therefore, the term "essentially" is used in this document to define the potential lack of completeness inherent in many biological and chemical phenomena.

Essential sequence homology. As used herein, the term "substantial homology" refers to a comparison between amino acid or nucleotide sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at the respective positions. Homologous residues may be the same residues. Otherwise, homologous residues may not be identical residues, and will have similar structural and/or functional characteristics. For example, as is well known to those skilled in the art, certain amino acids are commonly classified as "hydrophobic" or "hydrophilic" amino acids and/or having "polar" or "non-polar" side chains. The substitution of one amino acid for another of the same type can often be considered a "homologous" substitution. Typical classifications of amino acids are formulated as follows:

Alanine Ala A non-polar Neutral 1.8 Arginine Arg R Polar Positive -4.5 Asparagine Asn N Polar Neutral -3.5 Aspartic acid asp D Polar negative -3.5 Cysteine Cys C non-polar Neutral 2.5 Glutamic acid Glu E Polar negative -3.5 Glutamine Gln Q Polar Neutral -3.5 Glycine gly G non-polar Neutral -0.4 Histidine His H Polar positive -3.2 Isoleucine ile I non-polar Neutral 4.5 Leucine Leu L non-polar Neutral 3.8 Lysine Lys K Polar Positive -3.9 Methionine Met M non-polar Neutral 1.9 Phenylalanine Phe F non-polar Neutral 2.8 Proline Pro P non-polar Neutral -1.6 Serene Ser.. S Polar Neutral -0.8 Threonine Thr T Polar Neutral -0.7 tryptophan trp W non-polar Neutral -0.9 Tyrosine Tyr Y Polar Neutral -1.3 Valine Val V non-polar Neutral 4.2 Undefined amino acids 3 letter 1-letter Asparagine or aspartic acid asx B Glutamine or glutamic acid glx Z Leucine or Isoleucine Xle J Undefined or unknown amino acid xaa X

As is well known in the art, amino acid or nucleotide sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST and PSI-BLAST for amino acid sequences. An example of such programs is described in Altschul et al., 1990, J. Mol. Biol., 215(3):403-410; Altschul et al., 1996, Meth. Enzymology 266:460-480; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; Baxevanis et al, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al, (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs mentioned above usually indicate the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least , 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of their respective residues are homologous compared to the corresponding number of residues . In some embodiments, the relevant fragment is the entire sequence. In some embodiments, the relevant fragment is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least , 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225 at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more residues.

essential identity. As used herein, the term "substantial identity" refers to a comparison between amino acid or nucleotide sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain the same residues at the respective positions. As is well known in the art, amino acid or nucleotide sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST and PSI-BLAST for amino acid sequences. An example of such programs is described in Altschul, et al, 1990, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al, 1996, Methods in Enzymology 266:460-480; Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402; Baxevanis et al, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al, (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the programs mentioned above usually indicate the degree of identity. In some embodiments, two sequences are considered substantially identical if at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94%, 95%, 96%, 97 %, 98%, 99% or more of their respective residues are identical compared to the relevant fragment of the residues. In some embodiments, the corresponding fragment is the entire sequence. In some embodiments, the relevant fragment is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125 , 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more. In the context of a CDR, reference to "substantial identity" generally refers to a CDR having an amino acid sequence of at least 80%, preferably at least 85%, at least 90%, at least 95%, at least at least 98% or at least 99% identical to the sequence of the reference CDR.

Surface plasmon resonance. As used herein, the term refers to an optical phenomenon that allows the analysis of specific binding interactions in real time, for example by detecting changes in protein concentration in a biosensor array, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New -Jersey). For additional descriptions, see Jonsson, U. et al., 1993, Ann. Biol. Clin. 51:19-26; Jonsson, U. et al., 1991, Biotechniques 11:620-627; Johnsson, B. et al., 1995, J. Mol. Recognize. 8:125-131; and Johnnson, B. et al., 1991, Anal. Biochem. 198:268-277.

Therapeutic agent: As used herein, generally refers to any agent that produces the desired pharmacological effect when administered to the body. In some embodiments, an agent is considered a therapeutic agent if it demonstrates a statistically significant effect in the relevant population. In some embodiments, the respective population may be a population of model organisms. In some embodiments, the appropriate population may be defined by various criteria such as specific age group, gender, genetic background, pre-existing clinical conditions, and so on. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, reduce, inhibit, prevent, delay the onset, reduce the severity and/or reduce the incidence of one or more symptoms or features of a disease, disorder and/or condition. In some embodiments, a "therapeutic agent" is an agent that has been or must be approved by a government agency before it can be sold for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription for administration to humans.

Therapeutically effective amount: As used herein, is meant the amount that produces the desired effect for which it is administered. In some embodiments, this term refers to an amount that is sufficient when it is administered to a population suffering from or susceptible to a disease, disorder and/or condition in accordance with the therapeutic dosing regimen, for the treatment of the disease, disorder and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity and/or delays the onset of one or more symptoms of a disease, disorder, and/or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment in a particular individual. Rather, a therapeutically effective amount may be one that provides the specific pharmacological response sought in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, a reference to a therapeutically effective amount may be a reference to an amount measured in one or more specific tissues (e.g., tissue affected by a disease, disorder, or condition) or fluid (e.g., blood, saliva, serum, sweat, tears, urine, and etc.). Those skilled in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a single dose. In some embodiments, the therapeutically effective agent may be formulated and/or administered in multiple doses, for example, as part of a dosing regimen.

Transformation. As used herein, the term refers to any method by which exogenous DNA is introduced into a host cell. Transformation can occur naturally or artificially using a variety of methods well known in the art. Transformation may be based on any known method for introducing foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. In some embodiments, a particular transformation methodology is selected based on the transformed host cell and may include, but is not limited to, viral infection, electroporation, mating, lipofection. In some embodiments, the "transformed" cell is stably transformed in that the inserted DNA is capable of replication, either as an autonomously replicating plasmid or as part of the host's chromosome. In some embodiments, the transformed cell transiently expresses the introduced nucleic acid for a limited period of time.

Treatment: As used herein, the term "treatment" (also "treat" or "treat") in its broadest sense refers to any administration of a substance (e.g., the compositions of the present invention) that partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity and/or reduces the prevalence of one or more symptoms, signs and/or causes of a particular disease, disorder and/or condition. In some embodiments, such treatment may be administered to an entity that is not showing signs of the relevant disease, disorder, and/or condition and/or an entity that is only showing early signs of the disease, disorder, and/or condition. Alternatively, or additionally, in some embodiments, treatment may be administered to an entity that exhibits one or more established features of a relevant disease, disorder, and/or condition. In some embodiments, the treatment may be carried out on an object that has been diagnosed as suffering from an appropriate disease, disorder and/or condition. In some embodiments, the treatment may be carried out on the subject, which is known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the corresponding disease, disorder and/or condition.

Variant: As used herein, the term "variant" refers to an entity that shows significant structural identity with the reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, the variant is also functionally different from its reference object. In general, whether a particular entity should properly be considered a "variant" of the reference entity is based on its degree of structural identity with the reference entity. As will be understood by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a single chemical entity that shares one or more of these characteristic structural elements. As a few examples, a polypeptide may have a characteristic sequence element consisting of a plurality of amino acids having designated positions relative to each other in linear or three-dimensional space and/or contributing to a particular biological function, a nucleic acid may have a characteristic sequence element consisting of a plurality of nucleotide residues, having designated positions relative to another in linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical components (eg, carbohydrates, lipids, etc.) covalently linked to the polypeptide backbone. In some embodiments, the variant polypeptide shows a common sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95 %, 96%, 97% or 99%. Alternatively or additionally, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more biological activities of the reference polypeptide. In some embodiments, the variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, the variant polypeptide exhibits a reduction in the level of one or more biological activities compared to the reference polypeptide. In many embodiments, a polypeptide of interest is considered a "variant" of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to the amino acid sequence of the parent sequence, but with few sequence changes at specific positions. As a rule, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are replaced compared to the original sequence. In some embodiments, the variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the original sequence. Often, a variant has a very small number (eg, less than 5, 4, 3, 2, or 1) functional residues (ie, residues that are involved in a particular biological activity) replaced. In addition, a variant typically has no more than 5, 4, 3, 2, or 1 insertions or deletions, and often has no insertions or deletions compared to the original sequence. Moreover, any additions or deletions are typically less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6 and are typically less than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature. As will be appreciated by those skilled in the art, many variants of a particular polypeptide of interest will typically be found in nature, especially when the polypeptide of interest is an infectious agent polypeptide.

Vector: As used herein, the term refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. One type of vector is a "plasmid", which refers to a cyclic double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is the viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

Wild type: As used herein, the term "wild type" has its art-understood meaning, which refers to an entity having the structure and/or activity found in nature in "normal" (as opposed to mutant, variant, sick, changed, etc.) state or context. Those skilled in the art will appreciate that wild-type genes and polypeptides often exist in several different forms (eg, alleles).

Detailed description of some embodiments

The present invention is based in particular on the recognition that human antibody-based agents (e.g., human monoclonal antibodies) that have high specificity for CD19, in particular human CD19, can be made to preferentially bind to CD19 in a native format. (that is, the form expressed on the cell surface). In addition, such human antibody-based agents generally have superior in vivo properties compared to antibody agents that preferentially bind to recombinant or immobilized forms of CD19 (eg, plate-bound CD19). The present invention is also based on the recognition that human antibody agents developed from naive repertoires may, in some embodiments, not provide human antibody agents having specificity for clinically relevant epitopes. As one example, the present disclosure specifically demonstrates that the human anti-CD19 antibody agents described herein, in particular, a human antibody agent developed using an antibody library derived from sequences derived from a subject with an autoimmune disease, demonstrated binding to cell surface expressed CD19 but not to recombinant human CD19 in the context of a chimeric protein. Thus, the proposed human antibody agents can be characterized in some embodiments by high specificity for native human CD19 rather than non-native forms of human CD19, which potentially have little to no therapeutic value.

The present invention, inter alia, demonstrates the successful production of multispecific binding agents (eg, bispecific antibodies) that bind human CD19 expressed on the surface of several cancer cell lines and primary human B cells. In particular, the present invention mainly provides multispecific binding agents (eg, bispecific antibodies) that bind multiple cancer cell lines in a CD19-specific manner and mediate high efficiency in target cell lysis. Such multispecific binding agents are characterized by high specificity and efficiency in cytotoxicity assays.

The present invention also demonstrates the successful production of chimeric antigen receptors (CARs) using the present human antibody agents and subsequent expression in transduced T cells, resulting in engineered CAR-T cells (CAR-T). In particular, the present disclosure provides CAR-Ts that specifically recognize human CD19 expressed on the surface of various cancer cell lines. As described herein, the anti-CD19 CAR-Ts of the present invention exhibit effective target cell killing that in some embodiments is superior to CAR-Ts derived from components of other anti-CD19 antibodies. In addition, as described herein, the proposed anti-CD19 CAR-Ts are effective in eradicating tumors in animals implanted with human lymphoma xenografts. Thus, the present disclosure, in at least some embodiments, encompasses the development of human antibody agents having high specificity for native human CD19 for use in the diagnosis and treatment of cancer, and provides a source of antibody components for the production of multispecific binding agents and CAR-expressing agents. immune cells for use in humans.

CD19

Cluster 19 or CD19 is a transmembrane glycoprotein that is expressed on B cells, in particular a large number of B cell lines, with the exception of plasma cells. CD19 is also expressed on a number of malignant B cells. CD19 functions as a co-receptor on the surface of B cells in regulating the stimulation threshold for antigen-BCR interaction. CD19 also interacts with other cell surface molecules such as CD21, CD81 and CD82. Activation of CD19 (ie phosphorylation of the cytoplasmic domain) induces various signal transduction pathways involving various kinases (eg Src families) and leads to various processes in the immune cell. CD19 has been implicated in various cancers and autoimmune diseases. Indeed, CD19 is being intensively investigated as a target for the development of antibody-based therapeutics for cancer treatment (see e.g. Hammer, 2012, mAb 4:5, 571-577, Reichert, JM and E. Dhimolea, 2012, Drug Discovery Today 17 (17/18): 954-963 Suresh, T. et al., 2014, J. Hematol. 7:58); however, most of these antibody therapeutics are humanized and/or chimeric variants of mouse antibodies. Ideally, fully human antibody based agents are preferred for the treatment of human patients. The amino acid sequence of human CD19 is shown below (the signal sequence is underlined).

human CD19 polypeptide
(SEQ ID NO:290) MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR

Anti-CD19 antibody agents

Although anti-CD19 antibodies are known and have been shown to be therapeutic agents in the treatment of cancer and autoimmune disorders in humans, many of them are of murine origin. In this context, the use of human antibody agents is particularly preferred because it reduces the likelihood of an immune response against the administered antibody. Thus, the present invention provides fully human antibody-based agents (e.g., human monoclonal antibodies and fragments thereof) developed from human B cell libraries that exhibit high specificity for human CD19 and, in some embodiments, overcome the limitations associated with existing humanized and /or chimeric versions of non-human antibodies (eg, mouse).

The present invention relates to methods and compositions for the treatment of CD19 related diseases, disorders and conditions and associated cancers, based on the administration of anti-CD19 agents based on human antibodies (for example, monoclonal antibodies) and in some embodiments, multispecific binding agents (for example, bispecific antibodies) having a first antigen-binding site that binds human CD19 and a second antigen-binding site that binds an immune cell (eg, T cell).

The present invention specifically provides certain human antibody sequences, as well as certain affinity matured antibody sequences that bind human CD19 and in particular its native form (ie, expressed on the cell surface). For example, specifically exemplified herein are human antibody agents (e.g., full length antibodies, fragments thereof, single chain antibodies, bispecific antibodies, or other antibody agent formats as described herein or known in the art) that include polypeptides , containing the elements of the sequence described in this document.

Exemplary human antibody-based agents (e.g., scFv) that bind human CD19 are shown in Table 1 (light chain variable region sequences (DNA and amino acid) are shown in bold; heavy chain variable region sequences (DNA and amino acid) are shown in italics, linker sequences are shown as underlined text).

Table 1

In various embodiments, an anti-CD19 human antibody agent of the present invention consists of heavy and light chain variable regions, wherein the heavy chain variable region has a sequence that is at least about 50% identical (e.g., at least , about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%), the heavy chain variable region, which is represented in table 1.

In various embodiments, the anti-CD19 human antibody agent of the present invention consists of heavy and light chain variable regions, wherein the heavy chain variable region has a sequence that is substantially identical or identical to the heavy chain variable region as shown in Table 1.

In various embodiments, an anti-CD19 human antibody of the present invention is comprised of heavy and light chain variable regions, wherein the light chain variable region has a sequence that is at least about 50% (e.g., at least about 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to the light chain variable region shown in Table 1 .

In various embodiments, the anti-CD19 human antibody agent of the present invention consists of heavy and light chain variable regions, wherein the light chain variable region has a sequence that is substantially identical or identical to the light chain variable region as shown in Table 1.

In various embodiments, an anti-CD19 human antibody agent of the present invention is comprised of heavy and light chain variable regions, wherein the heavy chain variable region has a sequence that is at least about 50% (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%), identical to the heavy chain variable region, which is presented in Table 1, and in which the light chain variable region has a sequence that is at least about 50% identical (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the light chain variable region shown in Table 1.

In various embodiments, an anti-CD19 human antibody agent of the present invention is comprised of heavy and light chain variable regions, wherein the heavy chain variable region has a sequence that is substantially identical or identical to the heavy chain variable region as shown in Table 1, and wherein the light chain variable region has a sequence that is substantially identical or identical to the light chain variable region as shown in Table 1.

In various embodiments, the anti-CD19 human antibody agent of the present invention consists of heavy and light chain variable regions selected from the heavy and light chain variable region sequences shown in Table 1.

In various embodiments, the anti-CD19 human antibody agent of the present invention is or comprises a single chain variable fragment (scFv), wherein the scFv comprises a sequence that is at least about 50% identical (e.g., at least by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%), scFv, which is presented in table 1.

In various embodiments, the anti-CD19 human antibody agent of the present invention is or includes a single chain variable fragment (scFv), wherein the scFv comprises a sequence that is substantially identical or identical to scFv as shown in Table 1.

In various embodiments, the anti-CD19 human antibody agent of the present invention is or comprises a single chain variable fragment (scFv), the scFv being selected from Table 1.

The proposed antibodies, including antibodies and/or their characteristic parts, or nucleic acids encoding them, can be obtained by any available methods. For example, protocols for antibody production are described in Harlow and Lane, Antibodies: A Laboratory Manual (1988). Technologies for producing antibodies (eg, monoclonal antibodies and/or polyclonal antibodies) are well known in the art. It is understood that a wide range of animal species, including rabbits, mice, rats, hamsters, guinea pigs or goats, can be used to produce antisera. The choice of animal may be determined by ease of manipulation, cost, or amount of serum sought, as would be known to one of skill in the art. It is understood that antibody agents can also be produced transgenic by producing a mammal or plant that is transgenic for immunoglobulin heavy and light chain sequences of interest (eg, a rodent transgenic for human immunoglobulin heavy and light chain genes). In connection with transgenic production in mammals, antibodies can be obtained and extracted from the milk of goats, cows or other mammals (see, for example, US patents No. 5827690, 5 756 687, 5750172 and 5749557, incorporated herein by reference in its entirety). Typically, antibodies can be obtained from a mouse, rat, guinea pig, hamster, camel, llama, shark, or other suitable host. Alternatively, antibodies can be obtained from chickens producing IgY molecules (Schade et al., 1996, ALTEX 13 (5): 80-85). In some embodiments, antibodies suitable for the present invention are simian antibodies. For example, general methods for inducing therapeutically useful antibodies in baboons can be found, for example, in International Patent Application Publication No. 1991/11465 and in Losman et al., 1990, Int. J. Cancer 46: 310. In some embodiments, monoclonal antibodies can be obtained using hybridoma methods (Milstein and Cuello, 1983, Nature 305 (5934): 537-40). In some embodiments, monoclonal antibodies can also be obtained by recombinant methods (see, for example, US patent No. 4,166,452).

Many of the difficulties associated with generating monoclonal antibodies by immortalizing B cells can be overcome by constructing and expressing antibody fragments in E. coli (or yeast) using phage display. To provide high affinity recovery, monoclonal antibodies of combinatorial immunoglobulin libraries usually need to contain a large repertoire size. A typical strategy uses mRNA derived from lymphocytes or spleen cells of immunized mice to synthesize cDNA using reverse transcriptase. The heavy and light chain genes are amplified separately by PCR and ligated into phage cloning vectors. Get two different libraries: one includes the genes of the heavy chain, and the other includes the genes of the light chain. Libraries may be naive or may be semi-synthetic in which the heavy chain CDR3 is synthesized, with all amino acids (with the exception of cysteine) being equally likely to be present at any given position in the heavy chain CDR3 region. Phage DNA is isolated from each library, and the heavy and light chain sequences are ligated together and packaged to form a combinatorial library. Each phage includes a random heavy and light chain cDNA pair and, upon infection with E. coli, directs the expression of antibody chains in infected cells. To identify an antibody that recognizes an antigen of interest (eg, CD19), a phage library is seeded and the antibody molecules present in the plaques are transferred to filters. Filters are incubated with radiolabelled antigen and then washed to remove excess unbound ligand. A radioactive stain on the autoradiogram identifies a plaque that includes an antibody that binds the antigen. Alternatively, identification of an antibody that recognizes the antigen of interest (eg, CD19) can be achieved by iteratively binding the phage to an antigen that is bound to a solid support, such as beads or mammalian cells, then removing the unbound phage and eluting the specifically bound phage. . In such embodiments, the antigens are first biotinylated for immobilization, for example, to couple Dynabeads M-280 streptavidin. The phage library is incubated with cells, beads, or other solid support, and unbound phage is removed by washing. Phage antibody clones that bind the antigen of interest are selected and tested for further characterization.

After selection, positive scFv clones are tested for their binding to an antigen of interest expressed on the surface of living cells using indirect flow cytometry. Briefly, phage clones are incubated with cells (eg, engineered to express the antigen of interest, or those that naturally express the antigen) that either express or do not express the antigen. Cells are washed and then labeled with mouse monoclonal antibody against M13 coat protein. Cells are washed again and labeled with a fluorescent dye-conjugated secondary antibody (eg FITC-goat (Fab) ₂ against mouse IgG) prior to flow cytometry. Cloning and expression vectors that are useful for generating a human immunoglobulin phage library can be obtained, for example, from Stratagene Cloning Systems (La Jolla, Calif.).

A similar strategy can be used to generate high affinity scFv (see, for example, Vaughn et al., 1996, Nat. Biotechnol., 14: 309-314). A repertoire scFv library can be constructed by isolating V genes from non-immunized human donors using PCR primers corresponding to the well known V _H , Vκ and Vλ gene families. After amplification, the Vκ and Vλ pools are combined to form one pool. These fragments are ligated into a phagemid vector. The scFv linker (eg, (G ₄ S) _n ) is then ligated into the phagemid upstream of the V _L fragment (or upstream of the V _H fragment if desired). The V _H fragments and linker-V _L (or the V _L fragments and linker-V _H ) are amplified and assembled on the J _H region. The resulting V _H linker-V _L (or V _L linker-V _H ) fragments are ligated into a phagemid vector. The phagemid library can be sorted using filters as described above or using immunotubes (Nunc, Maxisorp). Similar results can be achieved by generating a library of combinatorial immunoglobulins from lymphocytes or spleen cells of immunized rabbits and by expressing the scFv constructs in P. pastoris (see, for example, Ridder et al., 1995, Biotechnology, 13: 255-260). In addition, once a suitable scFv is isolated, antibody fragments with higher binding affinity and slower dissociation rates can be generated by affinity maturation processes such as CDR3 mutagenesis and strand shuffling (see e.g. Jackson et al., 1998, Br J. Cancer, 78:181-188); Osbourn et al., 1996, Immunotechnology, 2: 181-196).

Another form of antibody fragment is a peptide encoding a single CDR. CDR peptides ("minimum recognition units") can be obtained by constructing genes encoding the CDRs of an antibody of interest. Such genes are obtained, for example, using polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al., 1991, Methods: Companion to Methods in Enzymology 2: 106; Courtenay-Luck, " Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering And Clinical Application, Ritter et al., eds., p. 166-179, Cambridge University Press 1995; and Ward et al., "Genetic Manipulation and Expression of Antibodies ," in Monoclonal Antibodies: Principles And Applications, Birch et al., eds., p. 137-185, Wiley-Liss, Inc. 1995).

Human antibodies can be generated using various methods, including phage display libraries (Hoogenboom et al., 1991, Mol. Immunol. 28(9):1027-37; Marks et al., 1991, J. Mol. Biol. 222 ( 3): 581-97) and the production of human monoclonal antibodies (Reisfeld and Sell, 1985, Cancer Surv. 4 (l): 271-90). Alternatively, the introduction of human Ig genes into transgenic animals in which endogenous Ig genes have been partially or completely inactivated can be used to synthesize human antibodies (see, for example, Fishwild et al., 1996, Nat. Biotechnol. 14(7) :845-51; Lonberg et al., 1994, Nature 368(6474):856-9; Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93; Taylor, L. D., et al., 1992, Nucl Acids Res 20:6287-95; Kellermann S-A., and Green L.L., 2002, Curr. Opin. Biotechnol. 13:593-7; Little, M. et al., 2000, Immunol. Today 21:364- 70; Murphy, A.J. et al., 2014, Proc. Natl. Acad. Sci. U.S.A. 111(14):5153-8). After infection, the formation of human antibodies is observed. In some embodiments, anti-human CD19 antibodies are produced by immunization of non-human animals engineered to produce human antibodies in response to challenge with human CD19 antigen.

Proposed agents based on a human antibody (including antibodies and/or characteristic parts) can be obtained, for example, using a host cell system designed to express a nucleic acid encoding an antibody of the invention. Alternatively or additionally, the proposed agents based on human antibodies can be partially or completely obtained by chemical synthesis (for example, using an automated peptide synthesizer or gene synthesis of nucleic acids encoding an antibody agent).

Human antibody agents of interest may be expressed using any suitable vector. Various vectors (eg, viral vectors) are known in the art; cells into which such vectors have been introduced (or progeny of such cells) can be cultured as is known in the art (eg, using continuous or fed-batch culture systems). In some embodiments, the cells may be genetically engineered; technologies for genetically engineered cells to express engineered polypeptides (e.g., antibody agent polypeptides as described herein) are well known in the art (see, e.g., Ausabel et al., eds., 1990, Current Protocols in Molecular Biology ( Wiley, New York).

In some embodiments, the subject human antibody agents may be purified, if necessary, using filtration, centrifugation, and/or various chromatographic techniques such as HPLC or affinity chromatography. In some embodiments, human antibody fragments of the subject agents are prepared by methods that include hydrolysis with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction.

In some embodiments, as described herein, the proposed human antibody agents may be or include, for example, a polyclonal antibody; a monoclonal antibody or antigen-binding fragment thereof; a modified antibody, such as an affinity matured antibody, a variant antibody (eg, having one or more amino acid substitutions compared to the reference antibody), or a fragment thereof (eg, Fab', Fab, F(ab') ₂ ); or a biosynthetic antibody, such as a single chain antibody, single domain antibody (DAB), Fv, single chain Fv (scFv), multispecific binding agents (eg, bispecific antibodies), chimeric antigen receptors (CAR), or the like.

It is understood that the subject human antibody agents may be designed, manufactured and/or purified in such a way as to improve the performance and/or activity of the antibody agents. For example, improved characteristics of the present antibody agents include, but are not limited to, increased stability, improved binding affinity and/or avidity, increased binding specificity, increased production, reduced aggregation, reduced non-specific binding, among others.

In some embodiments, the provided human antibody agents may contain one or more amino acid substitutions (e.g., in the framework region in the context of an immunoglobulin or fragment thereof (e.g., scFv)) that improve protein stability, antigen binding, expression level, or provide a site or site for conjugation of a therapeutic, diagnostic or detector.

In some embodiments, the provided human antibody agents are or include an immunoglobulin (i.e., antibody) that is a member of an antibody class (i.e., isotype) selected from the group consisting of IgG, IgM, IgA, IgD, IgE, or its fragment.

In some embodiments, the provided human antibody agents include human monoclonal antibodies that include a variant Fc region, wherein said variant Fc region contains at least one amino acid modification relative to a reference Fc region (or parent Fc region). In such embodiments, human antibody-based agents containing variant Fc regions may exhibit altered affinity for an Fc receptor (e.g., FcR), provided that said variant Fc regions do not have substitutions at positions that directly contact the Fc- receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions such as those described by Sondermann et al., 2000, Nature, 406: 267-273. Examples of positions in the Fc region that directly contact an Fc receptor such as FcR are amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C'/E loop). ) and amino acids 327-332 (loop F/G). In some embodiments, the proposed human antibody agents of the present invention containing variant Fc regions contain a modification of at least one residue that allows direct contact with FcR based on structural and crystallographic analysis.

In some embodiments, said human antibody agent is an anti-human CD19 monoclonal antibody having a variant Fc region with one or more amino acid modifications from the parent Fc region (or parent Fc region). Amino acid modifications in Fc regions to create variant Fc regions, which, for example, alter affinity for activation and/or inhibitory receptors, lead to improved effector function, such as, for example, antibody-dependent cytotoxicity associated with antibody (ADCC) and complementary dependent cytotoxicity (CDC), increase C1q binding affinity, decrease or abolish FcR binding, increase half-life, are known in the art (see, e.g., US Pat. 8,802,823, 8,802,820, 8,795,661, 8,753,629, 8,753,628, 8,735,547, 8,735,545, 8,734,791, 8,697,396, 8,546,543, 8,475,792, 8,399,618, 8,394,925, 8,388,955, 8,383,109, 8,367,805, 8,362,210, 8,338,574, 8,324,351, 8,318,907, 8,188,231, 8,124,731, 8,101,720, 8,093,359, 8,093,357, 8.088.376, 8.084.582, 8.039.592, 8.012.476, 7.799.900, 7.790.858 371.826, 7.364.731, 7.335.742, 7.332.581, 7.317.091, 7.297.775, 7.122.637, 7.083.784, 6.737.056, 6.538.124, 6.528.624 and 6.194.5).

In some embodiments, said human antibody agent is an anti-human CD19 monoclonal antibody having an Fc region with variant glycosylation compared to the original Fc region (eg, aglycosylated). In some embodiments, variant glycosylation results from expression in an engineered cell line (see, for example, Umana, P. et al., 1999, Nat. Biotechnol. 17(2):176-80; Yamane-Ohnuki, N. et al. , 2004, Biotechnol Bioeng 87(5):614-22, von Horsten, H. H. et al., 2010, Glycobiol 20(12):1607-18, Beck, A. et al., 2010, Expert Opin. Drug Discov 5(1):95-111 and U.S. Patent Nos. 8,080,415 and 8,084,222, all of which are incorporated herein by reference). In some embodiments, the present invention provides multispecific binding agents having an anti-human CD19 antibody component that includes a variant Fc region. In some embodiments, the proposed multispecific binding agent includes an antibody component that exhibits variant glycosylation (eg, is aglycosylated) compared to the parent antibody from which the component may be derived.

In some embodiments, the present invention provides and/or uses antibodies or antibody agents containing a variant Fc region (i.e., the Fc region includes one or more additions, deletions, and/or substitutions relative to a corresponding reference Fc) that are characterized in that an altered ( for example, increased and/or decreased) effector function and/or affinity for FcR is increased or decreased relative to the reference Fc. These variations are within the skill of the art.

Thus, inter alia, the present invention relates to antibody agents and multispecific binding agents (eg, antibody agents) containing variant Fc regions that bind with greater affinity to one or more FcRs. Such agents preferably mediate effector function more effectively, as described below. In some embodiments, the present invention relates to antibody agents and multispecific binding agents (eg, bispecific antibodies) containing a variant Fc region that binds with weaker affinity to one or more FcRs. The reduction or exclusion of effector function is desirable in some cases, for example, in the case of antibodies whose mechanism of action is associated with blocking or antagonism, but not with the destruction of cells bearing the target antigen. In addition, exclusion of effector function is desirable in some embodiments when creating bispecific antibodies, as discussed below. Reduction or exclusion of effector function would be desirable in cases of autoimmune disease where FcγR activation receptors in effector cells could be blocked (this type of function would be present in host cells). As a rule, increased effector function can be directed to the tumor and cells expressing foreign antigens; in some embodiments, the effector function may be directed to non-tumor cells.

Fc variants for use in accordance with the present invention may be combined with other Fc modifications, including, for example, modifications that alter effector function. The present invention includes combining an Fc variant as described herein with other Fc modifications to provide additive, synergistic or novel properties in Fc antibodies or chimeras. In some such embodiments, the Fc variants may enhance the phenotype of the modification with which they are combined. For example, if an Fc variant is combined with a mutant known to bind FcγRIIIA with higher affinity than a comparable molecule containing the wild-type Fc region, then combination with the mutant results in a fold increase in FcγRIIIA affinity.

In some embodiments, in accordance with the present invention, the Fc variants described herein are incorporated into an antibody or Fc chimera to produce an engineered agent that includes one or more Fc glycoforms (i.e., one or more Fc polypeptides to which one or more covalently attached several carbohydrates) to a molecule containing an Fc region, where the carbohydrate composition of the glycoform differs chemically from the glycoform of the original molecule containing the Fc region.

In some embodiments, a multispecific binding agent (e.g., an antibody agent) as described herein may include an Fc variant that exhibits a glycosylation variant and/or may be expressed in a glycosylation deficient cell line such that the Fc region of the agent is produced with loss of glycosylation compared to a corresponding reference Fc region (eg, the original Fc region), or an Fc region expressed in a cell line not deficient in glycosylation.

In some embodiments, the antibodies used in accordance with the present invention may have a modified glycosylation site relative to the corresponding control antibody that binds to an antigen of interest (eg, CD19), preferably without altering the functionality of the antibody, such as antigen binding activity. As used herein, "glycosylation sites" includes any specific amino acid sequence in an antibody to which an oligosaccharide (ie, carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach. Oligosaccharide side chains are usually linked to the antibody backbone via N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an aspartic residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxy amino acid, eg serine, threonine. For example, an Fc-glycoform that has lost certain oligosaccharides, including fucose and terminal N-acetylglucosamine, can be produced in special CHO cells and exhibit enhanced ADCC effector function.

In some embodiments, the present invention encompasses methods for modifying the carbohydrate content of an antibody, as described herein, by adding or removing a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included herein (see, for example, US Patent Nos. 6,218,149, 6,472,511, US Patent Publication Nos. 2003-0115614 A1, 2002-0028486 A1, International Patent Publication WO 2003/ 035835, European Patent No. 0 359 096 B1). In some embodiments, the present invention relates to methods for modifying the carbohydrate content of an antibody (or a corresponding portion or component thereof) by removing one or more endogenous carbohydrate fragments of the antibody.

Engineered glycoforms may be useful for a variety of purposes including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be produced by any method known to one of skill in the art, such as using engineered or variant expression strains by co-expression with one or more enzymes, such as N-acetylglucosaminyltransferase I (GnTI), by expressing a molecule containing an Fc region ( or a variant Fc region) in different organisms or cell lines from different organisms, or by modifying the carbohydrate(s) after the Fc region containing molecule has been expressed. Methods for producing engineered glycoforms are known in the art and include, but are not limited to, those described in Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278: 3466-3473; US Patent No. 6602684; US Patent Application No. 2003-0157108 A1, 2003-0003097 A1; International patent publications WO 2000/61739, WO 2001/292246, WO 2002/311140 and WO 2002/30954; POTILLEGENT™ Technology (Biowa, Inc. Princeton, NJ); Engineering technology of GLYCOMAB™ glycosylation (GLYCART biotechnology AG, Zurich, Switzerland). See, for example, Okazaki et al., 2004, JMB, 336: 1239-49; US Patent Publication No. 2003-0115614 A1; International Patent Publication No. WO 2000/061739.

Specific Exemplary Embodiments - Heavy and Light Chain Immunoglobulin Combinations

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 2 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 2. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 2. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 2 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 2.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO:4 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO:4. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO:4. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 4 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 4.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 6 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 6. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 6. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 6 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 6.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 8 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 8. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 8. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 8 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 8.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 10 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 10. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 10. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 10 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 10.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 12 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 12. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 12. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 12 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 12.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 14 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 14. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 14. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 14 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 14.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 16 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 16. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 16. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 16 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 16.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 18 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 18. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 18. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 18 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 18.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO:20 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO:20. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO:20. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 20 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 20.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 22 and/or an immunoglobulin light chain variable region as shown in SEQ ID NO: 22. In some embodiments, the immunoglobulin heavy chain variable region as shown in SEQ ID NO: 22. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 22 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 22.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 24 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 24. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 24. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 24 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 24.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 26 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 26. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 26. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 26 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 26.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 28 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 28. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 28. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 28 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 28.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 30 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 30. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 30. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 30 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 30.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 32 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 32. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 32. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 32 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 32.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 34 and/or an immunoglobulin light chain variable region as set forth in SEQ ID NO: 34. In some embodiments, the immunoglobulin heavy chain variable region as set forth in SEQ ID NO: 34. a human antibody is an antibody that includes an immunoglobulin heavy chain variable region as shown in SEQ ID NO: 34 and an immunoglobulin light chain variable region as shown in SEQ ID NO: 34.

Specific Illustrative Embodiments - Specific Amino Acid Substitutions

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region that includes one or more amino acid substitutions at any of amino acid positions 3, 12, 16, 17, 25, 26, 32, 63, 69, 97, 102, 106, 108, 109, 113, 116, 117 and combinations thereof. In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region that includes at least one and up to five amino acid substitutions in the framework and/or CDR region.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region as set forth in SEQ ID NO. selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34 and further includes one or more amino acid substitutions at any of the amino acid positions 3, 12, 16, 17, 25, 26, 32, 63, 69, 97, 102, 106, 108, 109, 113, 116, 117 and combinations thereof.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region that includes one or more substitutions selected from the group consisting of Q3R, K12E, E16G, S17F, S25A, G26A, Y32F, S63F, T69A, A97V , T102S, M106L, Y108N, D109E, Q113L, L116M, M117L and combinations thereof.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin heavy chain variable region that includes one or more substitutions compared to a control immunoglobulin variable heavy chain variable region, and the immunoglobulin heavy chain variable region has a sequence that is present in any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 and 72.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin light chain variable region that includes one or more amino acid substitutions at any of amino acid positions 10, 16, 25, 34, 52, 54, 68, 69, 72, 75, 93, 95 and combinations thereof. In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin light chain variable region that includes at least one and up to five amino acid substitutions in the framework region and/or CDR region.

In some embodiments, the human antibody agent of the present invention comprises an immunoglobulin light chain variable region as set forth in SEQ ID NO. selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34 and further includes one or more amino acids substitutions at any of amino acid positions 10, 16, 25, 34, 52, 54, 68, 69, 72, 75, 93, 95, and combinations thereof.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin light chain variable region that includes one or more substituents selected from the group consisting of V10M, K16E, S25N, V34I, D52N, L54Q, N68T, T69M, L72M, S75N , S93T, D95E, D95G and combinations thereof.

In some embodiments, a human antibody agent of the present invention comprises an immunoglobulin light chain variable region that includes one or more substitutions compared to a control immunoglobulin light chain variable region, and wherein the immunoglobulin light chain variable region has a sequence that is any of SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 and 72.

In some embodiments, a human antibody agent of the present invention comprises immunoglobulin heavy and light chain variable regions that together comprise at least one and up to five amino acid substitutions in the framework region and/or CDR region.

Among other things, the present disclosure demonstrates that the presence of such amino acid substitutions in a human anti-CD19 antibody agent of the present invention increases the binding affinity and/or specificity of the human antibody agent for human CD19 relative to the parent human antibody agent. That is, the introduction of such amino acid substitutions into a human anti-CD19 agent improves its binding affinity for CD19 and, in particular, for native CD19 (ie, expressed on the cell surface).

Those skilled in the art are aware of the various techniques known in the art for effecting such administration, or otherwise for preparing, preparing, or making, polypeptides containing such sequences. Illustrative technologies useful in this regard are presented, for example, in Green & Sambrook, Molecular Cloning: The Laboratory Manual, Cold Spring Harbor Laboratory Press 2012.

Specific Illustrative Embodiments - Specific CDRs

As is known in the art, monoclonal antibodies consist of two heavy chains and two light chains, each of which includes variable regions and constant regions. The heavy chain variable region typically includes three CDRs, herein referred to as HCDR1, HCDR2, and HCDR3, flanked by framework regions (see, e.g., William E. Paul, Fundamental Immunology (7th ed.), Lippincott Williams & Wilkins 2013). The light chain variable region typically includes three complementarity determining regions (CDRs), identified herein as LCDR1, LCDR2 and LCDR3, flanked by framework regions.

The present invention provides human antibody agents comprising the CDRs shown in Tables 2 (HCDR) and 3 (LCDR) in any possible combination. By way of non-limiting example, the present invention includes human antibody agents including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO: 183, respectively.

The production of antibodies having the CDRs and/or framework regions of interest is well known in the art and is described, for example, in Strohl & Strohl, Therapeutic Antibody Engineering, Woodhead Publishing Limited 2012.

table 2

Clone HCDR1 HCDR2 HDDR3 9 GFTFSSYA
(SEQ ID NO:73) ISGSGGST
(SEQ ID NO:74) ARYYYSRLDY
(SEQ ID NO:75) 23 GFTFSSYA
(SEQ ID NO:76) ISASGGST
(SEQ ID NO:77) ARYYLSQIDS
(SEQ ID NO:78) 31 GYTFTDYY
(SEQ ID NO:79) VDPEDGET
(SEQ ID NO:80) ATGIYSRPLGY
(SEQ ID NO:81) 35 GFTFSSYA
(SEQ ID NO:82) ISGSGGST
(SEQ ID NO:83) ARSDGKHFWQQYDA
(SEQ ID NO:84) 36 GFTVSSNY
(SEQ ID NO:85) ISGSGGST
(SEQ ID NO:86) ARMNIDY
(SEQ ID NO:87) 37 EYSFASYW
(SEQ ID NO:88) IDPSDSYT
(SEQ ID NO:89) ARPFQYDYGGYSDAFDI
(SEQ ID NO:90) 40 GGTFSSSS
(SEQ ID NO:91) IIPIVGTP
(SEQ ID NO:92) ARGGYRDYMDV
(SEQ ID NO:93) 43 GFTVSSNY
(SEQ ID NO:94) IYSGGST
(SEQ ID NO:95) ARGGFGAEFDY
(SEQ ID NO:96) 54 GFTVSSNY
(SEQ ID NO:97) IYSGGST
(SEQ ID NO:98) ARGGISDDYYGSGSYDN
(SEQ ID NO:99) 61 GFTVSSNY
(SEQ ID NO:100) IYSGGST
(SEQ ID NO:101) ARERGMGYAFDI
(SEQ ID NO:102) one GVSMSENY
(SEQ ID NO:103) AHYTGDT
(SEQ ID NO:104) ASYHPFNY
(SEQ ID NO:105) 2 GYTFNDFY
(SEQ ID NO:106) IDPEDGKT
(SEQ ID NO:107) TTDWGYSSSLREEDIWYDC
(SEQ ID NO:108) 3 GGTFSSYA
(SEQ ID NO:109) IIPIFGTA
(SEQ ID NO:110) ARDYGYGDYGDAFDI
(SEQ ID NO:111) four GYSFTSYW
(SEQ ID NO:112) IYPGDSDT
(SEQ ID NO:113) ARVVGTIYSMQYDV
(SEQ ID NO:114) 5 GYSFTSYW
(SEQ ID NO:115) IYPGDSDT
(SEQ ID NO:116) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:117) 6 GYSFTSYW
(SEQ ID NO:118) IYPGDSDT
(SEQ ID NO:119) ARWSSTWDSMYMDY
(SEQ ID NO:120) 7 GYSFTSYW
(SEQ ID NO:121) IYPGDSDT
(SEQ ID NO:122) ARVTYSMDSYYFDS
(SEQ ID NO:123) 5-1 GYSFTSYW
(SEQ ID NO:124) IYPGDSDT
(SEQ ID NO:125) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:126) 5-2 GYSFTSYW
(SEQ ID NO:127) IYPGDSDT
(SEQ ID NO:128) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:129) 5-3 GYSFTSYW
(SEQ ID NO:130) IYPGDSDT
(SEQ ID NO:131) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:132) 5-4 GYSFTSYW
(SEQ ID NO:133) IYPGDSDT
(SEQ ID NO:134) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:135) 5-5 GYSFTSYW
(SEQ ID NO:136) IYPGDSDT
(SEQ ID NO:137) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:138) 5-7 GYSFTSYW
(SEQ ID NO:139) IYPGDSDT
(SEQ ID NO:140) ARQVWGWQGGMNPRSNWWYNMDS
(SEQ ID NO:141) 5-9 GYSFTSYW
(SEQ ID NO:142) IYPGDSDT
(SEQ ID NO:143) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:144) 5-10 gysftsfw
(SEQ ID NO:145) IYPGDSDT
(SEQ ID NO:146) VRQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:147) 5-11 GYSFTSYW
(SEQ ID NO:148) IYPGDSDT
(SEQ ID NO:149) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:150) 5-13 GYSFTSYW
(SEQ ID NO:151) IYPGDSDT
(SEQ ID NO:152) ARQVWGWQGGMYPRSNWWYNLDS
(SEQ ID NO:153) 5-15 GYSFTSYW
(SEQ ID NO:154) IYPGDSDT
(SEQ ID NO:155) ARQVWGWQGGMYPRSNWWYNMDS
(SEQ ID NO:156) 6-1 GYSFTSYW
(SEQ ID NO:157) IYPGDSDT
(SEQ ID NO:158) ARWSSSWDSMYMDY
(SEQ ID NO:159) 6-2 GYSFTSYW
(SEQ ID NO:160) IYPGDSDT
(SEQ ID NO:161) ARWSSTWDSMYMEY
(SEQ ID NO:162) 6-4 GYSFTSYW
(SEQ ID NO:163) IYPGDSDT
(SEQ ID NO:164) ARWSSTWDSMYMDY
(SEQ ID NO:165) 6-5 GYSFTSYW
(SEQ ID NO:166) IYPGDSDT
(SEQ ID NO:167) ARWSSTWDSLYMDY
(SEQ ID NO:168) 6-6 GYSFTSYW
(SEQ ID NO:169) IYPGDSDT
(SEQ ID NO:170) ARWSSTWDSMYMDY
(SEQ ID NO:171) 6-7 AYSFTSYW
(SEQ ID NO:172) IYPGDSDT
(SEQ ID NO:173) ARWSSTWDSMYMDY
(SEQ ID NO:174) 6-8 GYSFTSYW
(SEQ ID NO:175) IYPGDSDT
(SEQ ID NO:176) ARWSSTWDSMYMDY
(SEQ ID NO:177) 6-9 GYSFTSYW
(SEQ ID NO:178) IYPGDSDT
(SEQ ID NO:179) ARWSSSWDSMYMDY
(SEQ ID NO:180)

Table 3

Clone LCDR1 LCDR2 LCDR3 09 SSNIGNNY DNN GTWDSSLSAGV (SEQ ID NO:181) (SEQ ID NO:182) (SEQ ID NO:183) 23 SSNIGNNY ENN GTWDSSLRAGV (SEQ ID NO:184) (SEQ ID NO:185) (SEQ ID NO:186) 31 SSNIGSNT SNN AAWDDSLNGHVV (SEQ ID NO:187) (SEQ ID NO:188) (SEQ ID NO:189) 35 SSNIGSHT SNN AAWDDSLNGYV (SEQ ID NO:190) (SEQ ID NO:191) (SEQ ID NO:192) 36 QGITNS AAS QHYLGTPYS (SEQ ID NO:193) (SEQ ID NO:194) (SEQ ID NO:195) 37 QSVSRF GVS QESYIPLT (SEQ ID NO:196) (SEQ ID NO:197) (SEQ ID NO:198) 40 QSLLDSNGFNS LGS MQSLQIPT (SEQ ID NO:199) (SEQ ID NO:200) (SEQ ID NO:201) 43 SSNIGSNY RNN AAWDDSLSGYV (SEQ ID NO:202) (SEQ ID NO:203) (SEQ ID NO:204) 54 KLGDKY QDN QAWDSSTEDV (SEQ ID NO:205) (SEQ ID NO:206) (SEQ ID NO:207) 61 QSISSY AAS QQSYSTPFT (SEQ ID NO:208) (SEQ ID NO:209) (SEQ ID NO:210) one QGIGSY PAS QQLNSL (SEQ ID NO:211) (SEQ ID NO:212) (SEQ ID NO:213) 2 SSNIGTKT SNY ALWDDSLDGYV (SEQ ID NO:214) (SEQ ID NO:215) (SEQ ID NO:216) 3 NIGSKS YDS QVWDSSSDHYV (SEQ ID NO:217) (SEQ ID NO:218) (SEQ ID NO:219) four NIGSKS DDS QVWDSSSDHYV (SEQ ID NO:220) (SEQ ID NO:221) (SEQ ID NO:222) 5 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:223) (SEQ ID NO:224) (SEQ ID NO:225) 6 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:226) (SEQ ID NO:227) (SEQ ID NO:228) 7 NIGSES YDS QVWNSSSDHRGV (SEQ ID NO:229) (SEQ ID NO:230) (SEQ ID NO:231) 5-1 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:232) (SEQ ID NO:233) (SEQ ID NO:234) 5-2 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:235) (SEQ ID NO:236) (SEQ ID NO:237) 5-3 NIGSKS DDS QVWDSSSEYVV (SEQ ID NO:238) (SEQ ID NO:239) (SEQ ID NO:240) 5-4 NIGSKS DDS QVWDSSSGYVV (SEQ ID NO:241) (SEQ ID NO:242) (SEQ ID NO:243) 5-5 NIGSKS DDS QVWDSTSDYVV (SEQ ID NO:244) (SEQ ID NO:245) (SEQ ID NO:246) 5-7 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:247) (SEQ ID NO:248) (SEQ ID NO:249) 5-9 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:250) (SEQ ID NO:251) (SEQ ID NO:252) 5-10 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:253) (SEQ ID NO:254) (SEQ ID NO:255) 5-11 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:256) (SEQ ID NO:257) (SEQ ID NO:258) 5-13 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:259) (SEQ ID NO:260) (SEQ ID NO:261) 5-15 NIGSKS DDS QVWDSSSDYVV (SEQ ID NO:262) (SEQ ID NO:263) (SEQ ID NO:264) 6-1 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:265) (SEQ ID NO:266) (SEQ ID NO:267) 6-2 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:268) (SEQ ID NO:269) (SEQ ID NO:270) 6-4 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:271) (SEQ ID NO:272) (SEQ ID NO:273) 6-5 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:274) (SEQ ID NO:275) (SEQ ID NO:276) 6-6 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:277) (SEQ ID NO:278) (SEQ ID NO:279) 6-7 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:280) (SEQ ID NO:281) (SEQ ID NO:282) 6-8 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:283) (SEQ ID NO:284) (SEQ ID NO:285) 6-9 SSNVGNNA YDD AAWDDSLNGPV (SEQ ID NO:286) (SEQ ID NO:287) (SEQ ID NO:288)

In various embodiments, the human antibody agent of the present invention comprises at least one of the HCDRs shown in Table 2 and/or at least one of the LCDRs shown in Table 3.

In various embodiments, the human antibody agent of the present invention comprises at least two of the HCDRs shown in Table 2 and/or at least two of the LCDRs shown in Table 3.

In various embodiments, the human antibody agent of the present invention includes any three HCDRs that are listed in Table 2 and/or any three LCDRs that are listed in Table 3.

In various embodiments, the human antibody agent of the present invention comprises a set of three HCDRs as shown in Table 2 and/or a corresponding set of three LCDRs as shown in Table 3.

In various embodiments, a human antibody agent of the present invention comprises three HCDRs, with each of the three HCDRs having a sequence that is at least about 50% identical (e.g., at least about 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), the three HCDRs shown in Table 2.

In various embodiments, a human antibody agent of the present invention comprises three LCDRs, with each of the three LCDRs having a sequence that is at least about 50% (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the three LCDRs shown in Table 3.

In various embodiments, a human antibody agent of the present invention comprises three HCDRs and three LCDRs, with each of the three HCDRs having a sequence that is at least about 50% (e.g., at least about 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the three HCDRs shown in Table 2, and each of the three LCDRs has a sequence , which is at least about 50% identical (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%), the three LCDRs shown in Table 3.

In various embodiments, a human antibody agent of the present invention comprises three HCDRs and/or three LCDRs, each of the HCDRs and LCDRs having a sequence that is substantially identical or identical to the HCDRs and LCDRs shown in Tables 2 and 3, respectively.

In various embodiments, the human antibody agent of the present invention is or includes an immunoglobulin (eg, a monoclonal antibody) that includes HCDRs and LCDRs selected from Tables 2 and 3, respectively.

Single chain Fv (scFv)

Single chain Fvs (scFvs) are widely known and used in the art. Single chain Fv is a chimeric protein of immunoglobulin heavy (V _H ) and light chain (V _L ) variable regions, sometimes connected by a short linker peptide (see, for example, see, for example, Benny KC Lo (ed.), Antibody Engineering - Methods and Protocols, Humana Press 2004, and sources cited therein).

The present invention provides single chain Fvs (scFvs) that specifically bind human CD19, and in some embodiments, the provided scFvs contain a linker sequence. In some embodiments, the V _H and V _L regions of scFv provided herein include a linker sequence that is SRGGGGSGGGGSGGGGSLEMA (SEQ ID NO: 289). In some embodiments, the linker sequence is a (G ₄ S) _n sequence, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater.

In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized in that it does not assume a rigid three-dimensional structure, but rather provides flexibility for the polypeptide (eg, the first and/or second linking components).

In some embodiments, the provided scFv polypeptide comprises a sequence that is shown in any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO : 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.

In various embodiments, the human antibody agent of the present invention is or includes a single chain variable fragment (scFv), the scFv comprising HCDR and LCDR selected from Tables 2 and 3, respectively.

In various embodiments, the human antibody agent of the present invention is or comprises a single chain variable fragment (scFv), wherein the scFv comprises a heavy and/or light chain variable region sequence as shown in Table 1.

In some embodiments, the proposed scFv polypeptide is conjugated to a therapeutic or detecting agent.

As known to those skilled in the art, the production of scFvs and their modification in accordance with the present invention are known in the art (see, for example, Benny K. C. Lo (ed.), Antibody Engineering - Methods and Protocols, Humana Press 2004, and references, cited there).

Multispecific binding agents

As known to those skilled in the art, a multispecific binding agent is a molecular entity or complex that includes binding components that specifically bind to two or more targets (eg, epitopes). Such multispecific binding agents find many uses in the art, including therapeutic uses. For example, as is known to those skilled in the art, multispecific binding agents have been designed to facilitate the killing of tumor cells by directing (or recruiting) cytotoxic T cells to the tumor site. Examples of tumor antigens include, but are not limited to, alpha fetoprotein (AFP), CA15-3, CA27-29, CA19-9, CA-125, calcretinin, carcinoembryonic antigen, CD34, CD99, CD117, chromogranin, cytokeratin, desmin, epithelial membrane protein (EMA), factor VIII, CD31 FL1, glial fibrillary acidic protein (GFAP), extensive cystic disease fluid protein-15 (GCDFP-15), HMB-45, human chorionic gonadotropin (hCG), inhibin, keratin, CD45 , lymphocytic marker, MART-1 (Melan-A), Myo Dl, muscle-specific actin (MSA), neurofilament, neurospecific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, protein S100, smooth muscle actin (SMA ), synaptophysin, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, and vimentin.

In some embodiments, multispecific binding agents for use in accordance with the present invention are bispecific binding agents. In many embodiments, such bispecific binding agents are capable of binding to tumor cells. In many embodiments, such bispecific binding agents are capable of binding to CD19 ⁺ cells or cells that express a CD19 polypeptide on the surface of a cell (eg, a tumor cell).

In some embodiments, multispecific binding agents (eg, bispecific binding agents) provided herein are or contain antibody components. Many technologies are known in the art for designing, constructing, and/or making multispecific binding agents containing antibody components.

For example, multispecific binding agents have been designed that either use the full structure of an immunoglobulin (eg, IgG), a single chain variable fragment (scFv), or combinations thereof. Bispecific binding agents consisting of two scFv units in tandem have been shown to be a clinically successful bispecific antibody format. In the case of antitumor immunotherapy, bispecific binding agents that contain two single-chain variable fragments (scFv) in tandem were designed such that the scFv that binds the tumor antigen was linked to the scFv that captures T cells by binding CD3 (e.g., CD3γ , CD3δ, CD3ε, CD3ζ, etc.). Thus, T cells are recruited to the tumor site in the hope that they can mediate the killing of tumor cells by the cytotoxic properties that certain T cells possess. An example has been given of such a bispecific binding agent that targets CD19 and CD3 for lymphoma (referred to as bispecific, T-cell recruiting or BiTE, for example, see Dreier et al., 2003, J. Immunol .170:4397-4402; Wolf, E. et al., 2005, Drug Discovery Today 10(18):1237-1244; Molhoj, M. et al. 2007, Mol. Immunol. 44:1935-43; Bargou et al., 2008, Science 321 :974-977; Baeuerle, P.A. and C. Reinhardt, 2009, Cancer Res. 69(12):4941-44; Hoffman, L.M and L. Gore, Frontiers in Oncol. 4 (article 63), 5 pp.), proved successful in preventing tumor growth in xenograft animal studies. In human studies, this bispecific binding agent has demonstrated objective tumor response, including five incomplete and two complete remissions.

Exemplary bispecific binding agents include those containing a first antibody component specific for a tumor antigen (eg, CD19) and a second antibody component specific for an immune cell (eg, T cell, NK cell, etc.). Bispecific binding agents can be obtained, for example, by combining heavy chains and/or light chains that recognize different epitopes of the same or different antigen. In some embodiments, via molecular function, the bispecific binding agent binds one antigen (or epitope) to one of its two binding arms (one V _H /V _L pair) and binds the other antigen (or epitope) to its second arm (the other V _H / V _L ). By this definition, a bispecific binding agent has two distinct antigen-binding arms (both in specificity and CDR sequences) and is monovalent for each antigen to which it binds.

In some embodiments, the bispecific binding agents of the present invention are characterized by the ability to simultaneously bind to two targets that have different structures. In some embodiments, the bispecific binding agents of the present invention have at least one component that specifically binds, for example, to a B cell, T cell, myeloid, plasma, or mast cell antigen or epitope, and at least one other a component that specifically binds CD19, in particular CD19 expressed on the cell surface.

In various embodiments, a bispecific binding agent (eg, a bispecific antibody) according to the present invention consists of a first binding component and a second binding component. In many embodiments, the first and second binding components of a bispecific binding agent, as described herein, each consist of antibody components with different characteristics. In many embodiments, antibody components are selected from Table 1.

In various embodiments, the bispecific binding agent of the present invention includes a first binding agent and a second binding agent. In various embodiments, a bispecific binding agent according to the present invention includes a first binding agent, a second binding agent, and a linker that is connected to both the first and the second binding agent (eg, located between the first and second binding components).

In various embodiments, the first and/or second binding components, as described herein, contain or are components of antibodies. In various embodiments, the first and/or second linking components, as described herein, contain a linker sequence or are linked through a linker sequence.

In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized in that it does not assume a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (eg, the first and/or second linking components). In some embodiments, a linker is used in the bispecific binding agent described herein based on particular properties conferred on the bispecific binding agent, such as, for example, reduced aggregation and/or improved stability. In some embodiments, the bispecific binding agent of the present invention includes a G ₄ S linker. In certain certain embodiments, the bispecific binding agent of the present invention includes a (G ₄ S) _n linker, where n is 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15 or more.

In some embodiments, the first and/or second binding components, as described herein, are or contain immunoglobulins (eg, IgG). In some embodiments, the binding components of the first and/or second binding components, as described herein, are antibody fragments (eg, scFv). In some embodiments, the first binding components, as described herein, are or contain antibody fragments, and the second binding components are or contain immunoglobulins. In some embodiments, the first binding components, as described herein, are or contain immunoglobulins, and the second binding components are or include antibody fragments. In some specific embodiments, the first binding components are immunoglobulins and the second binding components are antibody fragments. In certain specific embodiments, the first binding components are IgGs and the second binding components are scFvs.

In certain specific embodiments, the bispecific binding agent of the present invention includes a first and a second scFv. In certain specific embodiments, the first scFv is linked to the C-terminus of the second scFv. In certain specific embodiments, the second scFv is linked to the C-terminus of the first scFv. In certain specific embodiments, the scFvs are linked to each other via a linker sequence. In certain specific embodiments, the scFvs are linked to each other without linker sequences.

In some embodiments, the bispecific binding agent of the present invention includes two different heavy chains or heavy chain variable regions and two different light chains or light chain variable regions.

In some embodiments, the bispecific binding agent of the present invention has two specificities, one of which binds human CD19 and the other binds human CD3 (eg, CD3γ, CD3δ, CD3ε, CD3ζ, etc.) on a T cell.

In some embodiments, the bispecific binding agent of the present invention includes one or more sequences that are at least about 50% identical (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 95%, 96%, 97%, 98% or 99%), one or more of the sequences shown in Table 1.

In some embodiments, the bispecific binding agent of the present invention comprises one or more sequences that are substantially identical or identical to one or more of the sequences shown in Table 1.

In various embodiments, the first binding component of a bispecific binding agent as described herein comprises one or more HCDRs and one or more LCDRs, with each one or more HCDRs having a sequence that is at least 50% (e.g., 50 %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more), one or more HCDRs as shown in Table 2, and wherein each one or more HCDRs have a sequence that is at least 50% identical (e.g., 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of one or more LCDRs , which are presented in table 3.

In various embodiments, the first binding component of a bispecific binding agent as described herein comprises three HCDRs and three LCDRs, with each of the three HCDRs having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) HCDRs shown in Table 2 and each of the three LCDRs has a sequence that is at least 50% identical (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more), identical to the three LCDRs shown in Table 3.

In various embodiments, the first binding component of a bispecific binding agent, as described herein, includes a set of three HCDRs and a set of three LCDRs, with a set of every three HCDRs having a sequence that is at least 50% identical (e.g., 50 %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99 % or more), a set of three HCDRs as shown in Table 2, and each set of three LCDRs has a sequence that is at least 50% identical (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a set of three LCDRs that are shown in table 3.

In various embodiments, the first binding component of the bispecific binding agent as described herein includes a set of three HCDRs as shown in Table 2 and a set of three LCDRs as shown in Table 3.

In various embodiments, the first binding component of a bispecific binding agent as described herein comprises an antibody component having at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) antibody component, which is presented in table 1.

In various embodiments, the first binding component of the bispecific binding agent, as described herein, includes an antibody component having a sequence that is substantially identical or identical to the antibody component as shown in Table 1.

In various embodiments, the second binding component of the bispecific binding agent, as described herein, includes an antibody component that binds to a T cell. Exemplary T cell markers that can be used as targets for the second binding component of a bispecific binding agent as described herein include CD3 (e.g., CD3γ, CD3δ, CD3ε, CD3ζ, etc.), CD4, CD8 , CD28, OX40 (TNFRSF4 or CD134), GITR (TNFRSF18 or CD357), CD137 (TNFRSF9 or 4-1BB), CD27 (TNFRSF7), CD40L (TNFSF5 or CD154), HVEM (TNFRSF14 or CD270), etc. In some embodiments, the second binding component of the bispecific binding agent, as described herein, includes an antibody component that binds CD3. In some specific embodiments, the second binding component of the bispecific binding agent, as described herein, includes an antibody component that binds CD3ε. Exemplary anti-CD3 antibody components that can be used in the bispecific binding agents of the present invention are known in the art and can be found, for example, in Routledge et al., 1991, Eur. J. Immunol. 21:2717-25; Adair et al., 1994, Hum. Antibodies Hybridomas 5:41-7; Norman et al., 2000, Clin. transplant. 70(12):1707-12; Chatenoud, 2003, Nat. Rev. Immunol. 3(2):123-32; Cheadle, 2006, Curr. Opin. Mol. Ther. 8(1):62-8; Herold et al., 2009, Clin. Immunol. 132:166-73; Heiss and Murawa, 2010, Int. J. Cancer 127(9):2209-21; Keymeulen et al., 2010, Diabetologia, 53: 614-23; Silke and Gires, 2011, MAbs, 3(1):31-7; Cioffi, M. et al., 2012, Clin. Cancer Res. 18:465-474; Dean et al., 2012, Swiss Med. Wkly. 142:w13711; Labrijn et al., 2013, Proc. Nat. Acad. sci. U.S.A. 110(13):5145-50; Portell et al., 2013, Clin. Pharmacol. 5:5-11; US patent No. 712324, 7575923, 8 076 459, 8 1017222, which are all included in this description by reference in its entirety.

Otherwise, existing components, non-human (e.g., mouse) anti-CD3 antibodies, provide a valuable source of antibody components for use in the bispecific binding agents of the present invention (see, e.g., mouse OKT3 light chain accession number EMBL A22259.1 GL641464 and murine OKT3 heavy chain accession number EMBL A22261.1 GL21727144). While such non-human antibody components are useful, their humanized forms are preferred in human treatment because, among other things, it reduces the likelihood of immune reactions against the administered antibody (or bispecific binding agent). Methods for making humanized antibodies are generally known in the art.

Chimeric antigen receptors

The present invention also relates to chimeric antigen receptors containing a human antibody agent and/or a multispecific binding agent as described herein. CARs can be constructed by methods known in the art (see, for example, International Patent Application Publications Nos. 2012/079000, 2013/126726, 2015/177789 and 2015/080981; US Patent Application Publications No. 2012/0213783 A1, US Pat. Nos. 7,446,179 and 5912172, Milone, MC et al., 2009, Mol.Therapy 17(8):1453-64, Kochenderfer, J.N. et al., 2010, Blood 116(19):3875-86, 2011, N. Engl J. Med. 365(20):1937-9; Grupp, S. A. et al., 2013, N. Engl. J. Med. 368(16):1509-18; Romas, C. A. et al., 2014, Cancer J. 20(2):112-18, Singh, H. et al., 2014, Immunol Rev. 257(1):181-90, Fujiwara, H., 2014, Pharmaceuticals 7:1049-68, Maude, S.L. et al., 2015, Blood 125(26):4017-23, all of which are incorporated herein by reference). Immune effector cells (e.g., T cells, NK cells, etc.) can be engineered to express a chimeric antigen receptor that includes the antibody component described herein, resulting in antitumor immune effector cells that bypass mechanisms immune system surveillance used by cancer cells to prevent detection. In some embodiments, the present invention provides an immune cell that expresses a chimeric antigen receptor, the chimeric antigen receptor comprising one or more antigen-binding sites of a human antibody agent or multispecific binding agent as described herein. In some embodiments, the immune cells include T cells engineered to express the antigen-binding site of the human antibody agent described herein. In some embodiments, the antigen-binding site is or includes an antibody component as described herein.

In some embodiments, the immune cells include a chimeric antigen receptor that is human and/or human engineered (eg, includes one or more amino acid substitutions). In some embodiments, the chimeric antigen receptor includes one, two, three, four, five or more components (eg, antibody components, binding components, signaling components, etc.). In some embodiments, the chimeric antigen receptor includes a transmembrane component and/or an intracellular signaling component. In some embodiments, the chimeric antigen receptor includes components native to an immune cell from which said chimeric antigen receptor (eg, a native immune cell receptor) is expressed. In some embodiments, the chimeric antigen receptor comprises one or more transmembrane and/or one or more intracellular components of a T cell receptor (TCR) polypeptide.

The term "T cell receptor" or "TCR", as used herein, refers to a heterodimeric receptor consisting of αβ or γδ chains that pair on the surface of a T cell. Each α, β, γ, and δ chain consists of two Ig-like domains: a variable region (V), which provides for antigen recognition via CDRs in the TCR, followed by a constant region (C), which is anchored to the cell membrane via a linking peptide and a transmembrane (TM) region, which, in turn, connects to a cytoplasmic sequence/intracellular component. The TM region is associated with invariant subunits of the CD3 signaling apparatus. Each of the TCR V domains has three CDRs. These CDRs interact with a complex between an antigenic peptide associated with a protein encoded by the major histocompatibility complex (pMHC) (see, for example, Davis and Bjorkman, 1988, Nature 334: 395-402, Davis et al., 1998, Annu. Rev. Immunol. 16: ^523-44 ; Murphy, Kenneth P. Janeway's Immunobiology. 8th Edition. New York: Garland Science, Taylor & Francis Group, LLC, 2012. Print. Chapters 5 & 6). The TCR V domain and CDR sequences are generally very different from the V domain and CDR sequences of an antibody.

In some embodiments, the chimeric antigen receptor (CAR) is comprised of a heterodimer of the first and second polypeptides, wherein the first polypeptide comprises the anti-CD19 heavy chain variable region as described herein and the TCR γ chain TM region, and the second polypeptide comprises the light chain variable region. chains against CD19 as described herein, and the TM region of the TCR δ chain. In some embodiments, the first CAR heterodimer polypeptide comprises an anti-CD19 heavy chain variable region as described herein and a TCR δ chain TM region, and the second polypeptide comprises an anti-CD19 light chain variable region as described herein and a TM γ region TCR chains. In some embodiments, the proposed chimeric antigen receptor includes the TM region of TCR αβ chains. In some embodiments, the proposed chimeric antigen receptor includes the intracellular domain of the TCR. In specific embodiments, the proposed chimeric antigen receptor includes both the TM region and the intracellular TCR region.

In some embodiments, the components of the chimeric antigen receptor, as described herein, facilitate the binding of an immune cell to a CD19 ⁺ cell. In some embodiments, a chimeric antigen receptor, as described herein, includes an antibody component that is or is based on an antibody agent, as described herein. In some embodiments, the chimeric antigen receptor described herein comprises an antibody component that binds human CD19 and further comprises one or more cytoplasmic signaling domains in whole or in part and one or more co-stimulatory molecules in whole or in part. In some embodiments, the antibody component of a chimeric antigen receptor as described herein is a scFv; in certain specific embodiments, the scFv is selected from Table 1.

targets

Among other things, the present invention embraces the recognition that multispecific binding agents (and/or chimeric antigen receptors), and especially bispecific binding agents such as bispecific antibodies, are particularly useful and/or effective in facilitating cell killing. In particular, the present invention demonstrates that the activity of multispecific binding agents that specifically bind to both a target cell-associated epitope (e.g., CD19 on cancer cells) and a lymphocyte-associated epitope (e.g., T-cell surface protein) , may be an effective immunotherapy for B-cell disease and cancer.

For example, in some embodiments of the present invention, the multispecific binding agent specifically binds to a tumor cell associated epitope and to a T cell epitope. According to such embodiments, the multispecific binding agent may facilitate binding of the agent to one or both of the target epitopes and/or may enhance target T cell-mediated killing of the target tumor cell. Thus, for example, in some embodiments of the present invention, the chimeric antigen receptor binds CD19 on a cancer cell. In accordance with such embodiments, the immune cell can promote the destruction of the CD19 ⁺ cell after the interaction of the chimeric antigen receptor with its target epitope.

In some embodiments, the target cells to be killed include, for example, cells that express CD19 (eg, lymphoma or leukemia cells). Those skilled in the art will know suitable target epitopes on such cells to which it is desirable to bind multispecific binding agents, as described herein.

In some embodiments, lymphocyte cells that can mediate target cell killing as described herein include T cells (e.g., CD8 ⁺ T cells), natural killer (NK) cells, macrophages, granulocytes, and antibody-dependent cytotoxic cells. Those skilled in the art will be aware of the appropriate target epitopes on such lymphocytes to which it is desirable to bind the multispecific binding agents described herein. Such representative epitopes can be found on antigens such as, for example, the IgG Fc receptor (eg, FcγRIIB), CD1d, CD3 (eg, CD3γ, CD3δ, CD3ε, CD3ζ, etc.), CD4, CD7, CD8, CD13 , CD14, CD16, CD27, CD28, CD31, CD38, CD40L, CD56, CD68, MAC-l/MAC-3, IL-2Ra, OX40, GITR, HVEM, Ly49 and CD94, CD137.

Nucleotide construction and expression

Human antibody-based agents (eg, human monoclonal antibodies, scFv, etc.), multispecific binding agents (eg, bispecific antibodies), and chimeric antigen receptors as described herein can be generated from nucleic acid molecules using molecular -biological methods known in the art, Nucleic acid molecules are introduced into a vector that is capable of expressing chimeric proteins when introduced into an appropriate host cell. Suitable host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Any of the methods known to one of skill in the art for introducing DNA fragments into a vector can be used to construct expression vectors encoding the chimeric proteins of the present invention under the control of transcriptional/translational control signals. These methods may include in vitro recombinant DNA and synthetic methods and in vivo recombination (see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Harbor Laboratory, Current Protocols in Molecular Biology, Eds. Ausubel, et al, Greene Publ. Assoc., Wiley-Interscience, NY).

The expression of nucleic acid molecules according to the present invention can be regulated by a second nucleic acid sequence such that the molecule is expressed in a host transformed with the recombinant DNA molecule. For example, the expression of the nucleotide molecules of the invention may be controlled by a promoter and/or enhancer element, which are known in the art.

Nucleotide constructs include regions that encode multispecific binding proteins (or chimeric antigen receptors) derived from antibodies and/or antibody components. Typically, such multispecific binding proteins (or chimeric antigen receptors) will be derived from the V _H and/or V _L regions. Once antibodies exhibiting the desired binding and/or functional properties have been identified and selected, the variable regions of each antibody are isolated, amplified, cloned, and sequenced. Modifications may be made to the V _H and V _L nucleotide sequences, including additions of nucleotide sequences encoding amino acids and/or restriction sites, deletions of nucleotide sequences encoding amino acids, or substitutions of nucleotide sequences encoding amino acids. Antibodies and/or antibody components can be derived from human, non-human (eg, rodent), humanized, or chimeric antibodies.

The nucleotide constructs of the present invention are introduced into an expression vector or viral vector by methods known in the art and the nucleotide molecules are operably linked to an expression control sequence.

If necessary, the nucleotide sequences that encode human antibody agents, multispecific binding agents, and chimeric antigen receptors as described herein can be modified to include codons that are optimized for expression in a particular cell type or organism (e.g., see U.S. Patent Nos. 5,670,356 and 5,874,304). Codon-optimized sequences are synthetic sequences and preferably encode an identical polypeptide (or a biologically active fragment of a full-length polypeptide that has substantially the same activity as the full polypeptide) encoded by the parent non-codon-optimized polynucleotide. In some embodiments, the coding region of the genetic material encoding antibody components may include, in whole or in part, an altered sequence to optimize codon usage for a particular cell type (eg, eukaryotic or prokaryotic cell). For example, a coding sequence for a human heavy (or light) chain variable region, as described herein, can be optimized for expression in bacterial cells. Alternatively, the coding sequence may be optimized for expression in a mammalian cell (eg, CHO). Such a sequence can be described as a codon-optimized sequence.

The expression vector containing the nucleic acid molecule is transformed into a suitable host cell to provide the protein encoded by the nucleotide constructs. Examples of host cells include prokaryotes (eg E. coli) and eukaryotes (eg COS or CHO cell). Host cells transformed with the expression vector are grown under conditions to produce the human antibody agent, multispecific binding agent, or chimeric antigen receptor of the present invention, followed by recovery of the human monoclonal antibody, multispecific binding agent, or chimeric antigen receptor. In some embodiments, host cells include those that display a variant glycosylation pattern in one or more expressed polypeptides (see, for example, US Pat. Nos. 8,080,415 and 8,084,222).

The human antibody agents and/or multispecific binding agents of the present invention may be purified by any method that allows subsequent formation of a stable antibody or binding agent molecule. For example, without wishing to be bound by any particular theory, human antibody agents and/or multispecific binding agents can be isolated from cells either as soluble polypeptides or as inclusion bodies from which they can be quantitatively extracted. 8M guanidine hydrochloride and dialysis. Conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, or gel filtration can be used to further purify the human antibody agents and/or multispecific binding agents of the present invention. The human antibody agents and/or multispecific binding agents of the present invention can also be isolated from conditioned media after secretion from eukaryotic or prokaryotic cells.

Chimeric antigen receptors expressed on the surface of immune cells can be obtained by methods known in this field. Typically, an expression vector carrying a nucleotide sequence that encodes a chimeric antigen receptor polypeptide is transformed into a host cell for expression. As described above, such a host cell can be a cell line or primary human cell isolates, such as T cells from PBMC. The expression vector may also include a sequence that encodes a signal peptide and/or a transmembrane domain and/or a signal domain such that expression of the chimeric antigen receptor is directed to the cell membrane. Such signal peptides are known to those skilled in the art. An expression vector that encodes a chimeric antigen receptor polypeptide is introduced into the host cell by methods known in the art, which include, for example, DNA transfection, electroporation, transfection, and infection (eg, with a virus such as adenovirus or retrovirus).

In some embodiments, nucleotide molecules encoding a chimeric antigen receptor as described herein allow expression in a T cell, thereby allowing the production of chimeric antigen receptor (CAR-T) T cells. The nucleotide molecule encoding the chimeric antigen receptor may be included in one or more vectors (eg, a lentiviral or retroviral vector) for expression in an immune effector host cell, such as a T cell. Exemplary immune cells include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and regulatory T cells. Methods for producing nucleotide molecules encoding chimeric antigen receptors and T cells, including such chimeric antigen receptors, are known in the art (see, for example, Till, B. G. et al., 2008, Blood 112(6):2261-71; Brentjens , R. et al., 2010, Molecular Therapy 18(4):666-8, Morgan, R.A. et al., 2010, Molecular Therapy 18(4):843-51, Park, T.S. et al., 2011, Trends Biotechnol 29(11):550-7; Grupp, S.A. et al., 2013, N. Engl. J. Med. 368(16):1509-18; Han, E.Q. et al., 2013, J. Hematol. Oncol 6:47; WO 2012/079000, WO 2013/126726; and U.S. Patent Application Publication No. 2012/0213783).

Conjugates

Several technologies are known in the art for attaching or conjugating a therapeutic or detection agent to an antibody agent. Some attachment techniques include the use of a metal chelate complex using, for example, an organic chelating agent attached to the antibody, such as diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycuride-3, (see, for example, US Pat. Nos. 4,472,509 and 4,938,948). The proposed human antibody agents can also interact with the enzyme in the presence of a crosslinking agent such as glutaraldehyde or periodate. For example, conjugates with fluorescein markers are prepared in the presence of these crosslinkers or by reaction with isothiocyanate.

In some embodiments, a human antibody agent described herein is associated with a payload object. In some embodiments, the payload object is or includes a therapeutic agent; in some embodiments, the payload object is or includes a detecting agent.

Protecting groups

The human antibody agents (or multispecific binding agents or chimeric antigen receptors) of the present invention may be provided with protecting groups which are known in the art. Without wishing to be bound by any particular theory, the addition of a protecting group may, in some embodiments, improve one or more characteristics of the protected polypeptide or peptide, such as stability and/or efficacy. In some embodiments, the human antibody agents (or multispecific binding agents or chimeric antigen receptors) described herein may contain one or more (eg, one, two, three, four, five, or more) protecting groups. Protecting groups can be attached to the N- and/or C-terminal polypeptide or polypeptide chain of a human antibody agent (or multispecific binding agent or chimeric antigen receptor) as described herein. Protecting groups suitable for use in accordance with the present invention include those described in US Provisional Application No. 62/131, 128, which is incorporated herein by reference.

Therapeutic agents

Therapeutic agents may be or contain any class of chemical including, for example, proteins, carbohydrates, lipids, nucleic acids, small organic molecules, non-biological polymers, metals, ions, radioisotopes, and the like. In some embodiments, therapeutic agents for use in accordance with the present invention may have biological activity related to the treatment of one or more symptoms or causes of cancer. In some embodiments, therapeutic agents for use in accordance with the present invention may have biological activity related to the modulation of the immune system and/or to enhance T-cell mediated cytotoxicity. In some embodiments, therapeutic agents for use in accordance with the present invention have one or more other types of activities.

In some embodiments, the conjugated therapeutic agent is a radioisotope, drug conjugate, nanoparticle, immune toxin, or any other therapeutic payload.

Detecting agents

The detecting agent includes any part that can be detected using analysis, for example, due to its specific functional properties and/or chemical characteristics. Non-limiting examples of such agents include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands such as biotin.

Many detecting agents are known in the art, as well as systems for attaching them to antibodies (see, for example, US Pat. Nos. 5,021,236, 4,938,948, and 447,509). Examples of such detecting agents include paramagnetic ions, radioactive isotopes, fluorochromes, NMR detectables, X-ray imaging tools, and others. For example, in some embodiments, the paramagnetic ion is one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II) neodymium ( III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), tervium (III), dysprosium (III), holmium (III), erbium (III), lanthanum (III) gold (III ), lead (II) and/or bismuth (III).

The radioactive isotope may be one or more of actinium-225, astatine-211, bismuth-212, carbon-14, chromium-51, chlorine-36, cobalt-57, cobalt-58, copper-67, europium-152, gallium -67, hydrogen-3, iodine-123, iodine-124, iodine-125, iodine-131, indium-111, iron-59, lead-212, lutetium-177, phosphorus-32, radium-223, radium-224 , rhenium-186, rhenium-188, selenium-75, sulfur-35, technetium-99m, thorium-227, yttrium-90 and zirconium-89. Radioactively labeled agents based on human antibodies can be obtained in accordance with well known technologies in this field. For example, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite or an enzymatic oxidizing agent such as lactoperoxidase. The proposed human antibody agents can be labeled with technetium-99m by a ligand exchange process, for example, by reducing pertechnetate with tin solution, chelating the reduced technetium on a Sephadex column, and loading the antibody onto the column. In some embodiments, the subject human antibody agents are labeled using direct labeling techniques, for example, by incubating pertechnate, a reducing agent such as SNCl ₂ , a buffer solution such as sodium and potassium phthalate solution, and the antibody. In some embodiments, the proposed human antibody agents may be conjugated with intermediate functional groups. Intermediate functional groups are often used to bind radioisotopes, which exist as metal ions, to antibodies. Radioactive isotopes can be detected, for example, by dosimetry.

The fluorescent label may be or include one or more of Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3 , Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine and/ or Texas Red, among others.

In some embodiments, the conjugated detection agent is a diagnostic or imaging agent.

Screening, detection and therapeutic methods

The human antibody agents, multispecific binding agents, and/or chimeric antigen receptors of the present invention may also be used in in vitro or in vivo screening methods where it is desired to detect and/or measure one or more cell or cell activities (e.g., apoptosis or cell growth). Screening methods are well known in the art and include cell-free, cellular and animal assays. The in vitro assays may be the detection of a target molecule in either a solid state or a soluble state and may be carried out in a variety of ways known in the art, including the use of a label or a detectable group capable of identifying a human antibody agent, a multispecific binding agent, or a chimeric an antigen receptor that is bound to a target molecule (eg, a cell surface antigen). Detectable labels can be used in conjunction with assays using human antibody agents, multispecific binding agents, or chimeric antigen receptors of the present invention.

The ability of the human antibody agents, multispecific binding agents and/or chimeric antigen receptors of the present invention to exhibit high affinity and/or specificity for CD19 makes them therapeutically useful for effectively targeting CD19 expressing cells (eg, lymphoma or leukemia cells). Thus, in some embodiments, it may be desirable to increase the affinity of a human antibody agent or multispecific binding agent for one target antigen (e.g., CD19) rather than another target antigen that also binds with the multispecific binding agent (or Fc receptor in case of a human monoclonal antibody). For example, in the context of tumor killing, certain conditions may benefit from increased affinity for a tumor antigen (eg, CD19) but not for a cell surface antigen capable of mediating tumor killing (eg, T cells). Thus, it may be useful to increase the binding affinity and/or specificity of a human antibody agent, multispecific binding agent, or chimeric antigen receptor to a tumor antigen in a patient having a tumor that expresses a tumor antigen through the use of a human antibody agent, multispecific binding agent. or a chimeric antigen receptor as described herein.

The present invention relates to a human antibody agent, multispecific binding agent and/or chimeric antigen receptor as described herein as a therapeutic agent for the treatment of patients with a tumor that expresses an antigen that is able to bind to them. Such human antibody agents, multispecific binding agents, and/or chimeric antigen receptors may be used in a method of treating a human or animal organism, or in a diagnostic method.

In some embodiments, the proposed human antibody agent, multispecific binding agent, or chimeric antigen receptor is useful in medicine. In some embodiments, the human antibody agent, multispecific binding agent, or chimeric antigen receptor is useful (eg, as a prophylactic) in the treatment or prevention of a CD19-associated disease or cancer. In some embodiments, the inventive human antibody agent, multispecific binding agent, or chimeric antigen receptor is useful in treating patients who exhibit negative outcomes associated with or correlated with a CD20-associated disease or cancer (eg, ineffective internalization). In some embodiments, the inventive human antibody agent, multispecific binding agent, or chimeric antigen receptor is useful in therapeutic applications, for example, in individuals suffering from or associated with a CD19-associated disease or cancer.

Introduction

The present invention provides methods for administering an effective amount of a therapeutic active described herein (eg, a human antibody agent, multispecific binding agent, or chimeric antigen receptor) to a subject in need of treatment.

The human antibody-based agents, multispecific binding agents or chimeric antigen receptor described herein may be administered by various methods known in the art for therapeutic delivery of agents such as proteins or nucleic acids may be used for therapeutic delivery of an agent-based a human antibody, multispecific binding agent or chimeric antigen receptor or nucleic acid encoding a human antibody, multispecific binding agent or chimeric antigen receptor of the present invention for killing or inhibiting the growth of target cells in a subject, e.g., cell transfection, gene therapy, direct administration with a delivery carrier or a pharmaceutically acceptable carrier, indirect delivery by providing recombinant cells containing a nucleic acid encoding a human antibody, a multispecific binding agent, or a chimeric ant the gene receptor of the present invention.

Various delivery systems are known and can be used to administer the human antibody agent, multispecific binding agent, or chimeric antigen receptor of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing said antibody agent, multispecific binding agent, or chimeric antigen receptor, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), construction of a nucleic acid as part of a retroviral or other vector, etc. Routes of administration may be enteral or parenteral and include, but are not limited to, intravenous, subcutaneous, intramuscular, parenteral, transdermal, or transmucosal (eg, oral or nasal). In some embodiments, the human antibody agents, multispecific binding agents, or chimeric antigen receptors of the present invention are administered intravenously. In some embodiments, the human antibody agents, multispecific binding agents, or chimeric antigen receptors of the present invention are administered subcutaneously. In some embodiments, the human antibody agents, multispecific binding agents, or chimeric antigen receptors of the present invention are administered together with one or more biologically active agents.

Pharmaceutical compositions

The present invention also relates to pharmaceutical compositions containing the human antibody agents, multispecific binding agents or chimeric antigen receptors of the present invention and a pharmaceutically acceptable carrier or excipient. The composition, if necessary, may also contain one or more additional therapeutically active substances.

In some embodiments, pharmaceutical compositions containing a chimeric antigen receptor may be provided as a virus (eg, lentivirus or retrovirus) used to infect autologous T cells isolated from the subject. In such embodiments, autologous T cells are infected with a virus that has been engineered to express a chimeric antigen receptor, as described herein, and chimeric antigen receptor T cells are generated for reinfusion into a subject (eg, a human patient).

In some embodiments, pharmaceutical compositions containing a chimeric antigen receptor may be provided with a host cell (or populations thereof) expressing said chimeric antigen receptor and a pharmaceutical acceptor carrier. Examples of host cells include T cells or NK cells and may be autologous or allogeneic. Thus, host cells expressing a chimeric antigen receptor as described herein can be included in a pharmaceutical composition for administration to an object (eg, human). Such pharmaceutical compositions may contain a population of host cells expressing the same or different (two, three, four, etc.) chimeric antigen receptors described herein. Alternatively, a pharmaceutical composition comprising a host cell expressing the chimeric antigen receptor described herein may further comprise one or more additional pharmaceutically active agents or drugs such as, for example, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin. , cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In various embodiments, a pharmaceutical composition comprising the chimeric antigen receptor described herein is provided as a host cell expressing said chimeric antigen receptor or population thereof.

While the descriptions of pharmaceutical compositions provided herein are generally directed to pharmaceutical compositions that are suitable for ethical administration to humans, one of skill in the art will appreciate that such compositions are generally suitable for administration to animals of all kinds. Modification of pharmaceutical compositions suitable for administration to humans to make compositions suitable for administration to various animals is well understood and such modification can be designed and/or carried out by a skilled veterinary pharmacologist with simple, if necessary, experimentation.

The pharmaceutical compositions may be presented in a sterile injectable form (eg, a form suitable for subcutaneous injection or intravenous infusion). For example, in some embodiments, the pharmaceutical compositions are provided in a liquid dosage form that is suitable for injection. In some embodiments, the pharmaceutical compositions are provided as powders (eg, lyophilized and/or sterilized), optionally under vacuum, which are reconstituted with an aqueous diluent (eg, water, buffer, saline, etc.) prior to injection. In some embodiments, the pharmaceutical compositions are diluted and/or reconstituted in water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, the powder should be mixed gently with the aqueous diluent (eg without shaking).

The compositions of the pharmaceutical compositions described herein can be obtained by any method known or further developed in the field of pharmacology. In general, such preparation methods include the step of bringing the active ingredient to a diluent or other excipient and/or one or more other auxiliary ingredients, and then, if necessary and/or desired, shaping and/or packaging the product into the desired single or multi-dose unit.

The pharmaceutical composition of the present invention can be prepared, packaged and/or sold in bulk, as a unit dose and/or as multiple unit doses. As used herein, the term "unit dose" is a discrete amount of a pharmaceutical composition containing a given amount of an active ingredient. The amount of active ingredient is usually equal to the dose of the active ingredient that is administered to the subject and/or a convenient fraction of such a dose, such as, for example, half or one third of such a dose.

The relative amounts of active ingredient, pharmaceutically acceptable excipient and/or any additional ingredients in the pharmaceutical composition of the present invention will vary depending on the identity, size and/or condition of the subject being treated, as well as depending on the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w/w) of the active ingredient.

Pharmaceutical compositions may further comprise a pharmaceutically acceptable excipient which, as used herein, includes any and all solvents, dispersion media, diluents or other liquid carriers, dispersion or suspension agents, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, according to the particular dosage form sought. Remington's Science and Practice of Pharmacy, 21st edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in pharmaceutical compositions and known methods for their preparation. Except where any conventional excipient medium is incompatible with the substance or its derivatives, for example by inducing any undesirable biological effect or otherwise interacting in an adverse manner with any other component(s) of the pharmaceutical composition, its use is intended to be in within the scope of the present invention.

In some embodiments, the pharmaceutically acceptable excipient is at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, or 100% pure. In some embodiments, the excipient is approved for use in humans. In some embodiments, the excipient is approved for veterinary use. In some embodiments, the excipient is approved by the US Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient complies with the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia and/or International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surfactants and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents. , and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Auxiliary substances such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavors and/or flavors may be present in the composition according to the judgment of the formulator.

In some embodiments, the proposed pharmaceutical compositions contain one or more pharmaceutically acceptable excipients (eg, a preservative, an inert diluent, a dispersing agent, a surfactant and/or an emulsifier, a buffering agent, etc.). In some embodiments, the pharmaceutical compositions contain one or more preservatives. In some embodiments, the pharmaceutical compositions do not contain preservatives.

In some embodiments, the pharmaceutical compositions are provided in a form that can be refrigerated and/or frozen. In some embodiments, the pharmaceutical compositions are provided in a form that cannot be refrigerated and/or frozen. In some embodiments, reconstituted solutions and/or liquid dosage forms may be stored for a specified period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, a month, two month or more). In some embodiments, storage of antibody compositions longer than the specified time results in degradation of the antibody object.

Liquid dosage forms and/or reconstituted solutions may include suspended particles and/or discoloration prior to administration. In some embodiments, the solution should not be used if it is discolored or cloudy and/or if suspended particles remain after filtration.

General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington: Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005.

Sets

In addition, the present invention relates to a pharmaceutical package or kit containing one or more containers filled with at least one human antibody agent, multispecific binding agent (e.g., bispecific antibody), or chimeric antigen receptor (or immune effector cell with chimeric antigen receptor) as described herein. The kits can be used in any applicable method, including, for example, diagnostic. Optionally, such container(s) may be associated with a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, the notice of which reflects (a) approval by the authority for manufacture, use, or sale for administration to humans, ( b) directions for application, or both.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments, which are provided to illustrate the invention and are not intended to limit it. All patent and non-patent literature cited in this document is incorporated herein by reference in its entirety.

Examples

The following examples are provided to provide those skilled in the art with a description of how to make and use the methods and compositions of the invention and are not intended to limit the scope of what the authors consider their invention. Unless otherwise noted, temperatures are in degrees Celsius and pressures are at or near atmospheric pressure.

Example 1 Production and Selection of Human Antibody Agents Specific for Human CD19

This example demonstrates the production of human antibody-based agents specific for human CD19. In particular, this example demonstrates the production of human single chain variable fragments (scFv) that specifically bind human CD19 in native format. The human antibody agents described herein were developed using naive or semi-synthetic libraries of human antibodies derived from normal and/or autoimmune donors and selected based on high specificity for human CD19 by panning to human CD19 expressed on cell surface, in its native conformation. Thus, such human antibody-based agents provide a valuable source of antibody components for generating, among others, full-length IgGs, multispecific binding agents, and chimeric antigen receptors that may otherwise be removed from repertoires found in nature.

Briefly, an exemplary plan for the development of anti-CD19 antibody agents is shown in Table 4. The method began with the identification of human anti-CD19 and biologically active antibody agents from the Eureka ALPHA™ phage library. The best candidate antibody agents underwent affinity maturation to produce antibody agents with improved binding affinity and better cytotoxicity against cancer target cells.

A collection of human scFv antibody phage display libraries (diversity = 10×10 ¹⁰ ) constructed by Eureka Therapeutics and designated ALPHA™ phage libraries was used to select human antibody-based agents specific for human CD19. The ALPHA™ phage libraries included naive libraries consisting of fully naive human heavy and light chain repertoires and semi-synthetic libraries containing fully naive human light chain repertoires and semi-synthetic heavy chains with fully randomized heavy chain CDR3 regions. Naive antibody receptors were cloned from PBMCs and spleens of healthy donors, or from PBMCs of autoimmune disease donors (such as systemic lupus erythematosus and rheumatoid arthritis). The scFv libraries were used for panning against recombinant human CD19 chimeric ECD-Fc protein and human CD19 positive cells, including Raji and 3T3-CD19 cells (described below). For protein panning, the Fc chimeric protein was directly loaded onto 96-well plates and mixed with human scFv phage libraries. After extensive washing with PBS buffer, bound clones were eluted and used to challenge E. coli XL1-Blue. For cell panning, 3T3-CD19 or Raji cells were first mixed with human scFv phage libraries. After prolonged washing with PBS, cells with bound phage with scFv antibody were centrifuged. Bound clones were then eluted and used to infect E. coli XL1-Blue cells. Phage clones were expressed in bacteria and purified. Panning was performed for three to four rounds to enrich scFv phage clones that specifically bound the extracellular region of human CD19.

As shown in Table 5 (ALPHA™ Phage Display Summary), in cases where protein panning was included in the panning steps and ELISA binding against recombinant human CD19 chimeric protein ECD-Fc was used as a primary phage clone screening method, only one unique the antibody clone was identified as binding human CD19 on the cell surface after screening 762 phage clones from panning enriched pools. The authors then performed only cell panning and used a human CD19 positive cell line in the FACS assay as the primary screening method. Seven additional unique clones that bound human CD19 on the cell surface were identified from 690 clones screened using this panning campaign.

For ELISA assay, standard ELISA plates were coated with human CD19 ECD-Fc or 901-Fc negative control, respectively. Individual phage clones from panning-enriched phage display pools were incubated in coated plates. Phage clone binding was detected with HRP-conjugated anti-M13 antibodies and stained with HRP substrate. Absorbance was read at 450 nm. Several human CD19-ELISA-specific phage clones have been identified, and exemplary clones are shown in FIG. 1A. Among the screened 762 phage clones, 85 unique antibody clones were found to be specific for hCD19 as determined by ELISA.

ELISA-positive phage clones were then tested for cell surface CD19 binding by flow cytometry using the human CD19 positive Raji and 3T3-hCD19 cell lines and the human CD19 negative Jurkat and 3T3 cell lines (FIG. 1B). Cells were first stained with purified scFv phage clones followed by mixing with anti-mouse M13 monoclonal antibody and finally R-PE conjugated horse anti-mouse IgG (Vector Labs). Each stage of staining was carried out in the range of 30-60 min. on ice and cells were washed twice between stainings. One unique clone, 37, was identified as specifically binding human CD19 positive Raji cells and 3T3-CD19 rather than hCD19 negative Jurkat and 3T3 cells (FIG. 1B).

The results of the search after the second screen, in which phage clones were selected by cell panning, were only tested for binding to human CD19 directly on the cell surface without using an ELISA binding screen. As shown in FIG. 2, exemplary clones 4, 5 and 6 showed specific binding to Raji cells, while clone 7 also showed some detectable binding to Raji-CD19 knockout cells.

As shown in this example, the efficiency of phage panning of anti-human CD19 antibody was very low because of the 1452 individual phage clones identified by primary screens, only eight specific and sequence-unique clones were identified. One clone (37) was derived from a naive autoimmune disease library and seven other clones were derived from naive or semi-synthetic human antibody libraries derived from normal donor B cells.

Table 4

Stage Methodology Primary panning with the ALPHA™ phage library Protein and cell panning screening of phage clones by ELISA and/or FACs Clone characterization Destruction of targeted cancer cells Human B cell binding Affinity maturation Creation of an affinity matured phage library cell panning screening of phage clones with FACs Clone characterization K _D measurement Destruction of targeted cancer cells

Table 5

Protein/cell panning cell panning Number of clones
(ELISA screening) 762 Number of clones
(FACS screening) 690 Clones that bind CD19 on the cell surface one Clones that bind CD19 on the cell surface 7

Example 2 Preparation of Bispecific Antibody Constructs Using Anti-human CD19 Agents

This example demonstrates the construction of multispecific binding agents using human scFv fragments specific for human CD19. In particular, this example specifically demonstrates the construction of bispecific antibodies having a first antigen-binding site that binds native human CD19 (expressed on the cell surface) and a second antigen-binding site that binds CD3 on T cells. Thus, this example illustrates that in some embodiments, using multispecific binding agents that contain antibody components as described herein, T cells can be directed to kill target cells that express human CD19.

Bispecific antibodies were generated using scFv sequences from phage clones specific for human CD19. Bispecific antibodies were constructed using a single chain format containing the V _L -V _H scFv sequence of a human CD19-specific phage clone at the N-terminus and a mouse monoclonal anti-human CD3ε scFv at the C-terminus (e.g., see Brischwein, K. et al., Mol. Immunol. 43:1129-1143, 2006). DNA fragments encoding anti-human CD19 scFv and anti-human CD3ε scFv were synthesized by Genewiz or Genscript and subcloned into the pQD-T mammalian expression vector (Eureka Therapeutics, Inc.) using standard recombinant DNA technology. A hexahistamine label was inserted at the C-terminus for purification and detection. HEK293 cells were transfected with a bispecific antibody expression vector and cultured for seven days to obtain a bispecific antibody. Bispecific antibodies were purified from supernatants of HEK293 cells using a HisTrap HP column (GE healthcare) with an AKTA FPLC system. The HEK293 cell culture was purified and loaded onto a low imidazole (20 mM) column and then high imidazole (500 mM) isocratic elution buffer was used to elute the bound bispecific antibodies. The molecular weights of the purified anti-human CD19 bispecific antibodies were measured under non-reducing conditions by gel electrophoresis. Protein (4 μg) was mixed with 2.5 μl of NuPAGE LDS sample buffer (Life Technologies, NP0008) and made up to 10 μl with deionized water. The samples were heated to 70°C for 10 minutes and applied to the gels. Gel electrophoresis was carried out at 180 V for 1 hour. Bands (~55 kDa) corresponding to bispecific antibodies were observed as the main species on the gel (Fig. 3).

Taken together, this example demonstrates the successful construction of bispecific molecules having a first antigen-binding site that is specific for human CD19 expressed on the cell surface (i.e., in native format) and a second antigen-binding site that is specific for CD3. Further characterization of such bispecific molecules is described in the following examples.

Example 3 Characterization of anti-human CD19 bispecific antibodies

This example demonstrates the CD19 binding profile for selected bispecific molecules. In particular, this example specifically describes the binding profile of selected bispecific antibody molecules for recombinant human CD19 (ie, human CD19 ECD-Fc) in solution.

Binding to recombinant human CD19 ECD-Fc chimeric protein. Phage clones that were identified as binders specific for human CD19 cell surface were tested for binding to recombinant chimeric human CD19 ECD-Fc protein in solution using Fortebio Octet. Biotinylated human CD19 ECD-Fc chimeric proteins (5 μg/mL) were loaded onto a biosensor with streptavidin. After washing off excess antigen, the bispecific antibodies were tested at 10 μg/ml in PBS association and dissociation buffer. None of the clones tested that recognized cell surface expressed human CD19 showed binding to the recombinant human CD19 ECD-Fc chimeric protein, including clone 37, which was an ELISA positive clone. In FIG. 4 shows binding data of the hCD19 octet of exemplary clones and BL19, a comparative anti-CD19 bispecific antibody. Thus, the authors suggested that the recombinant hCD19 protein may have a different structural conformation compared to native human CD19 (ie, on the cell surface) as well as human CD19 bound to the ELISA plate. Coating an ELISA plate with recombinant human CD19 can generate certain conformation(s) that mimic native hCD19, but with extremely low efficiency. Only one of the 85 antibody clones specific for human CD19 according to ELISA showed specificity for cell surface human CD19. Cell panning with cells expressing human CD19 produced more positive antibody clones that were specific for human CD19 expressed on the cell surface.

Binding to primary human B cells. Human B cells were tested for binding to anti-CD19 antibodies by co-staining human PBMCs with PerCP-conjugated anti-human CD20, APC-labeled anti-human CD3, and anti-CD19 bispecific antibodies. After a round of brief washing with PBS buffer, FITC-labeled anti-His tag antibody was added to the mixture as a secondary antibody to detect the bispecific antibody. For flow cytometry analysis, human B cells were selected above the threshold for positive CD20 staining and negative CD3 staining (see inset in Figure 5A). Bispecific antibodies against CD19 were evaluated by their ability to recognize human CD19 expressed on these CD20 ⁺ CD3 ^- cells.

The level of anti-CD19 binding to CD20 ⁺ CD3 ^- cells was measured using the average fluorescence intensity (MFI) in the FITC channel. As shown in Table 6, the negative control bispecific antibody (NC-ET901) did not bind human B cells. In contrast, all anti-human CD19 bispecific clones tested recognized human B cells and showed positive binding signals.

Analysis for destruction by T-cells. Tumor cytotoxicity was analyzed using an LDH cytotoxicity assay (Promega). Human T cells (AllCells) or Ficoll-purified whole blood cells (Pacific Blood Centers) were activated and expanded with CD3/CD28 "Dynabeads" (Invitrogen) according to the manufacturer's specifications. Activated T cells were cultured and maintained in RPMI1640 medium with 10% FBS plus 100 U/ml IL-2 and used within 7-14 days after activation. T cells accounted for >99% CD3⁺according to the results FACS analysis. Activated T cells and target cells were co-cultured in a 5:1 ratio with bispecific antibodies for 16 hours. Cytotoxicity was determined by measuring LDH activity in culture supernatants.

As shown in FIG. 5B, bispecific antibody molecules based on clones 4, 5, and 6, at 0.2 μg/ml, effectively mediate the killing of cancer cells in a human CD19-specific manner. In addition, a bispecific antibody molecule based on clone 37 showed some detectable killing against CD19 negative cell lines (Jurkat and THP-1). In the context of scFv, clone 37 did not show binding to Jurkat cells according to flow cytometry (FIG. 1B).

Table 6

CD19xCD3
Bispecific antibody clone Average fluorescence intensity
(MFI) in the FITC channel four 545 5 1271 6 2195 7 1880 37 2083 NC-ET901 144

(NC-ET901: control)

Example 4 Affinity maturation of anti-human CD19 antibody agents

This example demonstrates the affinity maturation of anti-human CD19 antibody agents. In particular, this example specifically demonstrates the generation of a range of antibody variants by incorporating random mutations into selected anti-human CD19 antibody agents (clones 4, 5 and 6) followed by screening and characterization of the antibody variants.

Obtaining variant phage libraries. DNA encoding anti-human CD19 scFv was subjected to random mutagenesis using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies) as described by the manufacturer. After mutagenesis, the DNA sequences were cloned into an scFv-expressing phagemid vector to create phage libraries of variant antibodies. Mutation libraries were generated for each individual clone specific for human CD19. Each mutation library contained about 5 × 10 ⁸ unique phage clones. On average, variant clones have two nucleotide mutations compared to the original anti-human CD19 clones, ranging from 1 to 4 nucleotide mutations per scFv sequence. Three libraries were built on the original clones 4, 5 and 6 (table 7). Exemplary mutations in the V _H and V _L regions of selected affinity matured anti-human CD19 antibody agents compared to the corresponding parental clones are shown in Table 8.

Individual phage clones from the panning-enriched phage pools, termed variant clones, were tested for improved cell surface binding to human CD19 compared to their respective parental clones. As shown in FIG. 6A, several variant phage clones specifically recognize human CD19 positive cancer cell lines, including Raji and Jeko-1 cells. None of the clones showed binding to human CD19 negative cell lines such as Raji-CD19 k.o. or Jurkat. Notably, multiple clones showed increased binding to Raji and Jeko-1 cells compared to their respective parental clones.

A competitive cell binding assay was performed to compare the binding affinity of the variant clones compared to the original clones (FIG. 6B). Briefly, variant phage clones were mixed with Raji cells that were pre-incubated with their respective parental bispecific antibody clone, followed by staining with mouse anti-M13 antibody and PE-conjugated anti-mouse antibody.

Clone variants 5-1, 5-3, 5-4, 5-9, 5-13 and 6-1 showed improved target binding compared to their parent clones. In the presence of the original bispecific antibody clone, these phage clones exhibit increased binding to Raji cells compared to the original phage clones, clone 5 or 6. A more accurate measurement of antibody binding affinity was obtained by flow cytometry titration of the bispecific antibody (see Table 9 below) .

Table 7

original clone Name of the phage library Library size Mutation frequency (per 1000 bp) four ET190-4AM 3.25E+08 4.0 5 ET190-5AM 4.50E+08 3.1 6 ET190-6AM 5.70E+08 3.3

Table 8

Clone V _L V _H FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 5-1 S75N 5-2 T69M 5-3 D52N D95E 5-4 V10M D95G 5-5 S93T S17F T69A 5-7 N68T Y108N 5-9 S63F 5-10 Q3R Y32F A97V 5-11 K16E 5-13 M117L 5-15 L72M S25A 6-1 T102S L116M 6-2 S25N V34I K12E D109E 6-4 E16G 6-5 L54Q M106L 6-6 S25N 6-7 G26A Q113L 6-8 L54Q 6-9 T102S

Example 5 Generation and characterization of bispecific antibody molecules based on anti-human CD19 antibody variants

This example demonstrates the construction of multispecific binding agents using various human scFv fragments specific for human CD19. In particular, this example specifically demonstrates the construction of bispecific antibodies having a first antigen-binding site that binds human CD19 in native format (expressed on the cell surface) and a second antigen-binding site that binds CD3 on T cells. Thus, this example illustrates that, in some embodiments, using multispecific binding agents that contain the antibody components described herein, T cells can be targeted to kill target cells that express human CD19.

Obtaining bispecific antibodies of the variant clone. Bispecific antibodies derived from variant affinity enhanced antibody clones were generated as described in Example 2. Most of the clones showed single bands on SDS-PAGE with >90% purity (FIG. 7A).

Determination of binding affinity for variant bispecific antibodies. Neither the original antibodies nor their derivatives (variant clones) recognize recombinant human CD19, although they all bind human CD19 expressed on the cell surface. Thus, the authors determined the relative binding affinity of the variant clones compared to the original antibodies using flow cytometry antibody titrations using human CD19 positive cancer cells (Table 9).

Clones of bispecific antibodies in serially diluted concentrations were mixed with the same number of Raji cells. The EC ₅₀ and apparent K _D of the antibody were calculated based on flow cytometry binding signals. As shown in Table 9, many of the variant clones based on the original clone 5, with the exception of clone 5-4, show slightly increased affinity (lower EC ₅₀ and apparent K _D ). For variant clones based on parent clone 6, clones 6-1 and 6-4 also showed increased affinity (lower EC ₅₀ and apparent K _D ) compared to their respective parent clone.

Analysis of T-cell cytotoxicity. Tumor cytotoxicity was determined using an LDH cytotoxicity assay (Promega). Human T cells were activated and expanded with CD3/CD28 "Dynabeads" (Invitrogen) as described by the manufacturer. Activated T cells were cultured and maintained in RPMI1640 medium with 10% FBS plus 100 U/ml IL-2 and used within 7-14 days after activation. T cells were >99% CD3 ⁺ by FACS analysis. Activated T cells and target cells were co-cultured at a ratio of 5:1 with bispecific antibodies for 16 hours. Cytotoxicity was determined by measuring LDH activity in culture supernatants (FIG. 7B).

As shown in FIG. 8, the variant clones tested retained the original binding specificity of their parent antibodies. For the cytotoxicity assay shown in FIG. 11, exemplary variant clones were tested as well as clone 37 (used as a positive control bispecific antibody since this clone showed detectable non-specific cytotoxicity against human CD19 negative cells, Raji-CD19 k.o., Jurkat and THP-1). The multiple clone variants tested demonstrated equivalent or increased cell killing efficiency against one or both of the human CD19 positive cell lines (Raji and Jeko-1) compared to their respective parental clones.

Table 9

Clone EC ₅₀ (mcg/ml) apparent K _D (nM) #5 0.101 1.9 5-1 0.097 1.9 5-3 0.081 1.6 5-4 0.124 2.4 5-9 0.075 1.4 5-10 0.097 1.9 5-13 0.089 1.7 #6 0.214 4.1 6-1 0.116 2.2 6-2 4.02 77.3 6-4 0.156 3.0 6-7 0.242 4.7 NC-ET901 undetectable -

(NC-ET901: control)

Example 6 Characterization of T cells expressing chimeric antigen receptors (CAR-T) against human CD19

This example demonstrates the construction of chimeric antigen receptors (CARs) using the anti-human CD19 antibody agents described in the previous examples. In particular, this example specifically demonstrates a CAR construct that includes the antigen-binding site of a human anti-CD19 agent and is expressed on the surface of T cells. In addition, T cell-expressed CARs have been used in cytotoxicity assays against human lymphoma xenograft models. Thus, this example illustrates that, in some embodiments, the use of CARs that include an antigen binding site from the human antibody agents described herein are useful in killing target cells that express human CD19 (eg, lymphomas).

In vitro cytotoxicity of human CD19-transformed T cells

Lentiviruses containing chimeric antigen receptors (CARs) specific for human CD19 were produced by transfection of 293T cells with CAR vectors. Human T cells were used for transduction after one day of stimulation with CD3/CD28 beads (Dynabeads®, Invitrogen) in the presence of IL-2 at 100 U/ml. Concentrated lentiviruses were applied to T cells in retronectin-coated 6-well plates (Takara) for 72 hours. Functional evaluation of transduced T cells (CD19/CAR-T cells) was performed using an LDH cytotoxicity assay. Effector-target ratios were 5:1. Representative results are shown in Fig. eight.

As shown in FIG. 8, three CARs based on variant antibody clones (clones 5-3, 5-9, and 5-13) demonstrated significantly increased cancer cell killing against the Raji and Jeko-1 cell lines compared to the parent clone 5. the target used in this experiment was 5:1. The killing specificity was confirmed to be CD19-specific, as evidenced by the low killing rate of human CD19-negative cells (Raji-CD19 k.o. and Jurkat). In addition, T cells expressing CARs generated using the human antibody-based agents described herein demonstrated similar or higher cell killing efficiency than CAR-Ts generated with other anti-CD19 antibodies (BL19, FMC63) .

In a similar experiment, anti-human CD19 CAR-T (described above) was tested using a large panel of CD19 positive and negative cancer cell lines. Briefly, primary T cells were sham-transduced (Sham-transduced) or transduced with selected CARs encoding anti-CD19 antibodies. Transduced T cells were analyzed by FACS using an anti-myc antibody to detect a myc tag in the extracellular domain of the CAR constructs.

The results showed that anti-CD19 CAR-T cells derived from the human antibody agents described above have a transduction efficiency of ~80% (FIG. 9, top). The ability of CAR-T cells to specifically kill CD19-expressing cancer cells was tested at a 5:1 effector-target ratio. As shown in FIG. 9 (bottom), CAR-T cells expressing both tested anti-CD19 clones specifically killed CD19 ⁺ cells (Raji, CA46, JeKo-1 and Daudi) but not CD19 cells (Raji-CD19 ^- KO, Jurkat, THP-1, HeLa, MDA-MB-231, MCF-7, SK-Hep-1 and HepG2).

In another similar experiment, CAR-Ts derived from the selected human antibody agents described herein and non-human (e.g. murine) antibodies were tested using a panel of CD19 positive and negative cancer cell lines as described above. . Briefly, primary T cells were sham-transduced (Sham-transduced) or transduced with selected CARs encoding the anti-human CD19 scFv described herein (CAR-T 5, CAR-T 5-9, or CAR-T 5-13) or CARs encoding an anti-human CD19 scFv that has variable regions of sequences from the mouse anti-human CD19 mAb FMC63 (Zola, H. et al., 1991, Immunol. Cell Biol. 69(Pt 6):411-22). The mouse anti-CD19 ScFv sequence below is in the format V _L -linker-V _H . Transduced T cells were analyzed by FACs as described above.

mouse anti-CD19 scFv
(SEQ ID NO:296) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

The CDR sequences of the mouse anti-CD19 scFv shown in SEQ ID NO: 296 are:

LC-CDR1 QDISKY (SEQ ID NO:297)

LC-CDR2 HTS (SEQ ID NO:298)

LC-CDR3 QQGNTLPYT (SEQ ID NO:299)

HC-CDR1 GVSLPDYG (SEQ ID NO:300)

HC-CDR2 IWGSETT (SEQ ID NO:301)

HC-CDR3 AKHYYYGGSYAMDY (SEQ ID NO:302)

The results showed that anti-CD19 CAR-T cells derived from human scFv clone 5 and two affinity matured variants (5-9, 5-13) demonstrated higher transduction efficiency and significantly higher CD19-specific lysis of cancer cells. cells than anti-CD19 CAR T cells derived from mouse anti-CD19 antibody (FIG. 15).

In another experiment, an approximate cytokine release profile was determined by activated human CD19 CAR-T cells. Mock-transduced T cells (mock-transduced) or selected anti-CD19 CAR-T (clone 5-3 shown) were co-incubated with target cells. The release of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-γ and TNF-α to the medium after in vitro killing was measured using the Magpix multiplex system (Luminex) with assays Bio-plex Pro Human Cytokine 8-plex Assays (BioRad). Cytokine concentrations were determined using a known standard curve, after subtracting release from media, target cells only, and clone 5-3 transduced T cells only. Representative results are shown in Fig. ten.

As shown in FIG. 10, cytokine release was only detected when anti-CD19 CAR-T cells were co-incubated with CD19 ⁺ cells (Raji and CA46) but not CD19 cells (Raji ^- CD19 ko and Jurkat). Mock-transduced T cells co-cultured with CD19 ⁺ cancer cells released only minimal amounts of cytokines. Similarly, the cytokine release profile of activated human CD19-CAR T cells based on clone 5-13 showed cytokine release (IL-2, GM-CSF, IFN-γ, and TNF-α) only when anti-CD19 CAR -T - cells were co-incubated with CD19 ⁺ cells (Raji and JeKo-1), but not with CD19 ^- cells (Raji-CD19 ko). In addition, sham-transduced T cells co-cultured with CD19 ⁺ cancer cells released either minimal amounts of cytokines or no release at all.

In vivo efficacy of CD19 CAR-T cells in human lymphoma xenografts. The in vivo antitumor activity of an exemplary CAR-T cell (based on clone 5-9) was tested in a NOD SCID gamma (NSG) mouse model of CD19-positive human lymphoma xenograft. Briefly, Raji-luc-GFP cells were purchased from Comparative Biosciences, Inc. (Sunnyville, CA 94085) and cultured in RPMI medium + 10% FBS and 1% L-glutamine at 37°C in a humidified atmosphere with 5% CO ₂ . Raji-luc-GFP cells were derived from a CD19-positive Burkitt's lymphoma cell line, Raji, after stable transfection with dual reporter genes encoding firefly luciferase (luc) and green fluorescent protein, resulting in cells that can be traced in vivo using bioluminescent visualization. NSG mice were purchased from Jackson Laboratories (Bar Harbor, Maine, USA 04609) and acclimated at least 7 days prior to the experiment. Raji-luc-GFP cells were resuspended in PBS and implanted intravenously (iv) into NSG mice via the tail vein at 1×10 ⁶ cells/100 μl/mouse. Five days after implantation, the animals were imaged using the Xenogen IVIS imaging system to assess tumor mass. Mice were randomized based on photon emission into three groups (n = 6 mice per group) with an average photon emission of 6.7 × 10 ⁵ photons: (i) no treatment, (ii) sham-transduced (non-transduced activated human T cells from same donor as CAR-T cells) and (iii) 5-9 CAR-T. Animals were treated intravenously with sham-transduced or 5-9 CAR-T clone cells immediately after randomization at a dose of 10 ⁷ T cells per mouse (including 6-8 x 10 ⁶ CAR + T cells per dose for group (iii) ), once every two weeks for 3 doses. Animals were closely monitored after dosing. Bioluminescent imaging using the Xenogen IVIS system was performed once a week. Animals with the following conditions were euthanized and recorded as "conditional death": (i) weight loss of more than 25% of the original body weight and (ii) limb paralysis that affects the mobility of the mouse. Representative results are shown in Fig. eleven.

Both sham-transduced cells and CAR-T 5-9 cells were well tolerated at the current dose and schedule (FIG. 11). No dosing-related side effects were observed throughout the study. Mock-transduced T cell treatment retarded the growth of Raji-luc-GFP tumors (p < 0.05), however, CAR-T 5-9 treatment significantly inhibited tumor growth (p < 0.05, Fig. 11, right) .

In a similar experiment, the in vivo antitumor activity of another exemplary CAR-T cell (based on clone 5-13) was tested in a CD19-positive human lymphoma (Raji) xenograft model in NSG mice. Experiments were performed as described above for clone 5-9, except that each mouse was injected with only one dose of anti-CD19 CAR transduced T cells (5 x 10 ⁶ CAR ⁺ T cells based on clone 5-13) . Representative results are shown in Fig. 13.

As shown in FIG. 13A, while tumors grew rapidly in mice from the vehicle and mock-transduced groups, NSG mice injected with T cells that were transduced with anti-CD19 CAR 5-13 cells showed inhibition of tumor growth. Indeed, a significant difference in tumor growth between treatment groups was found as early as day 11 (p<0.0001, Dunnett's tests, Fig. 13B). In addition, NSG mice injected with T cells that are transduced with anti-CD19 CAR 5-13 showed tumor regression even 45 days after implantation.

35 days after tumor implantation, three representative mice from the group injected with anti-CD19 CAR 5-13 transformed T cells were revaccinated with implantation of 5 x 10 ⁵ Raji lymphoma cells in order to identify, maintain and maintain whether anti-CD19-CAR-transduced T cells have the ability to respond to an antigen (CD19). Naive NSG mice (i.e., mice not implanted with Raji lymphoma cells or not previously treated with T cells, n = 2) were implanted with 5 x 10 ⁵ Raji lymphoma cells one day after injection of 1 x 10 ⁶ mock-transduced T -cells (injected on day 34) as a control. The authors hypothesized that such mock-transduced T cells would mimic the low circulating state of T cells in mice prior to implantation of Raji lymphoma cells. Tumor burden in each group was measured using luciferase activity (as described above). Representative results are shown in Fig. 13C.

As shown in FIG. 13C, tumors grew rapidly in the sham-transduced cell treatment group. In contrast, prior treatment with anti-CD19 CAR 5-13 transduced T cells prevented tumor growth after re-infection with Raji lymphoma cells (p<0.01). Thus, these data indicate that CD19-CAR-transformed T cells persist in these mice even 35 days after implantation and retain the ability to respond to CD19 and kill CD19 ⁺ tumor cells, thereby conferring protection against tumor recurrence. loads.

In another similar experiment, the in vivo antitumor activity of an exemplary CAR-T cell (based on clone 5-13) was tested in a CD19-positive human leukemia xenograft model in NSG mice (NALM). NALM-6-luc-GFP cells (a courtesy gift from Dr. Eric Smith of Memorial Sloan-Kettering Cancer Center) were derived from the CD19-positive acute lymphoblastic leukemia cell line NALM-6 after stable transfection with double reporter genes encoding firefly luciferase (luc ) and green fluorescent protein (GFP), resulting in cells being tracked in vivo using bioluminescent imaging.

Briefly, NALM-6-luc-GFP was cultured in RPMI + + 10% FBS at 37°C in a humidified atmosphere with 5% CO ₂ . NSG mice were purchased from Jackson Laboratories (Bar Harbor, Maine, US 04609) and acclimated at least three days prior to experiments. NALM-6-luc-GFP cells were resuspended in phosphate buffered saline (PBS) and intravenously (iv) implanted in female NSG mice (n = 20, 6-8 weeks) at 5 x 10 ⁵ cells/100 μl/mouse through an injection in the tail vein. Four days after implantation, the animals were imaged using the Xenogen IVIS imaging system to assess tumor burden. NSG mice were randomized based on photon emission into three groups: (i) vehicle (PBS, n=6); (ii) 10 ⁷ mock-transduced human T cells (n=6); and (iii) 5x10 ⁶ clones 5-13 of anti-CD19 CAR transduced T cells (CAR-T 5-13, n=8). Animals were closely monitored after implantation and dosing with T cells or vehicle as described above. Representative results are shown in Fig. fourteen.

As shown in FIG. 14, Panels A and B, vehicle and mock-transduced cell treatment groups, demonstrated rapid growth progression of NALM-6 tumors. In contrast, treatment with anti-CD19 CAR-T cells (based on clone 5-13) significantly (p<0.0001) inhibited and even reversed tumor growth as early as day 11. These data show that CAR-T 5-13 cells targeted and lysed CD19 ⁺ NALM-6 tumors in vivo, demonstrating the efficacy of the anti-CD19 antibodies provided herein in an exemplary CAR format to inhibit tumor growth during many models of cancer.

Taken together, this example demonstrates the successful production and use of T cells expressing a chimeric antigen receptor (CAR) based on human antibody-based anti-CD19 agents prepared according to the previous examples. In addition, such anti-CD19 CAR-T cells have been shown to efficiently kill various cancer cell lines in a CD19-specific manner. Thus, the human antibody-based agents described herein can be used to treat and diagnose various B-cell diseases and pak, in particular those characterized by CD19 expression.

Ways

The Raji, Jeko-1, Jurkat, THP-1, and NIH/3T3 cell lines were obtained from the American Type Culture Collection. Raji is a CD19 positive Burkitt's lymphoma cell line (ATCC CCL-86). Jeko-1 is a CD19 positive mantle cell lymphoma cell line (ATCC CRL-3006). Jurkat is a human lymphocyte cell line derived from T-cell leukemia and is CD19 negative. THP-1, an acute monocytic leukemia cell line, is also CD19 negative (ATCC TIB-202). NIH/3T3 is a mouse embryonic fibroblast cell line (ATCC CRL-1658). The Raji-CD19 knockout cell line (Raji-CD19 ko) was generated by CD19 knockout using the commercial CRISPR-Cas9 vector (Origene). Raji-CD19 ko cells were confirmed to be CD19 negative by sequencing and FACS (FIG. 12) using a commercial mouse anti-human CD19 antibody (Biolegend). 3T3-CD19 was generated by transducing an NIH/3T3 cell line with a mammalian human CD19 expression vector (hCD19), resulting in high levels of hCD19 expression on the cell surface (FIG. 16). Other cell lines tested in CAR-T cytotoxicity assays such as CA46, Daudi, Hela, HepG2, SK-Hep1, MDA-MB-231 and MCF7 were also purchased from ATCC. Cell lines were cultured in RPMI 1640 or DMEM supplemented with 10% FBS at 37°C/5% CO ₂ .

A mouse monoclonal antibody against M13 phage was purchased from GE (Cat 27-9421-01). PE-labeled anti-mouse IgG antibody was purchased from Vector labs (Cat EI-2007). PE-conjugated mouse anti-hCD19 antibody was purchased from Biolegend (Cat 302208). 901-Fc and NC-ET901 bispecific antibody, which are negative control antibodies and have the format of human IgG1 and bispecific antibody, respectively, were obtained from Eureka Therapeutics, Inc. The antibody does not recognize the human antigen. A FITC-labeled anti-His antibody was purchased from Thermo Fisher Scientific (Cat MA1-81891) to detect a bispecific antibody that has a 6xHis tag at the C-terminus. PerCP-conjugated anti-human CD20 antibody was purchased from BD (9Cat 302208) to detect human B cells.

The recombinant human CD19 ECD-Fc fusion protein includes an extracellular domain of human CD19 fused to human IgG1 Fc, lacks a C _H 1 domain, and has a hinge region. HEK293 cells (Invitrogen) were transfected with a vector encoding the human CD19 ECD-Fc fusion protein using Lipofectamine 3000 (Invitrogen). The chimeric protein was purified from culture supernatants by protein A affinity chromatography.

Specific Aspects Listed

The present invention also provides the following numbered aspects:

1. A human antibody agent that specifically binds human CD19, wherein the human antibody agent comprises one or more heavy chain CDRs, each of which has a sequence that is at least 95% identical to one or more heavy chain CDRs selected from Table 2 and/or one or more light chain CDRs each having a sequence that is at least 95% identical to one or more light chain CDRs selected from Table 3.

2. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3, which are shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO: 183.

3. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 as set forth in SEQ ID NO: 184, SEQ ID NO: 185, and SEQ ID NO: 186.

4. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO: 189.

5. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192.

6. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195.

7. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of the light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO: 198.

8. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 as set forth in SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201.

9. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 202, SEQ ID NO: 203, and SEQ ID NO: 204.

10. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207.

11. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1. , a heavy chain CDR2 and a heavy chain CDR3 of those shown in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, each having a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 208, SEQ ID NO: 209, and SEQ ID NO: 210.

12. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO: 213.

13. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each of the light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216.

14. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1. , heavy chain CDR2 and heavy chain CDR3 of SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111, and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each of the light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219.

15. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114, and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO: 222.

16. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, and light chain CDR1, light chain CDR2 and light chain CDR3, each of which has a sequence that is at least 95% identical to each of the light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 223, SEQ ID NO: 224, and SEQ ID NO: 225.

17. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1. , heavy chain CDR2 and heavy chain CDR3 of those shown in SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120, and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 226, SEQ ID NO: 227, and SEQ ID NO: 228.

18. The human antibody agent of aspect 1, wherein the human antibody agent comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each having a sequence that is at least 95% identical to each heavy chain CDR1. , heavy chain CDR2 and heavy chain CDR3 of SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123, and light chain CDR1, light chain CDR2 and light chain CDR3, each having a sequence that is at least 95% identical to each light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NO: 229, SEQ ID NO: 230, and SEQ ID NO: 231.

19. The human antibody agent of aspect 2, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, and a light chain CDR of SEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183.

20. The human antibody agent of aspect 3, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, and a light chain CDR of SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186.

21. The human antibody agent of aspect 4, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81 and a light chain CDR of SEQ ID NO: 187, SEQ ID NO: 188 and SEQ ID NO: 189.

22. The human antibody agent of aspect 5, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, and a light chain CDR of SEQ ID NO: 190, SEQ ID NO: 191 and SEQ ID NO: 192.

23 The human antibody agent of aspect 6, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, and a light chain CDR of SEQ ID NO: 193, SEQ ID NO: 194 and SEQ ID NO: 195.

24. The human antibody agent of aspect 7, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90 and a light chain CDR of SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198.

25. The human antibody agent of aspect 8, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, and a light chain CDR of SEQ ID NO: 199, SEQ ID NO: 200 and SEQ ID NO 201.

26. The human antibody agent of aspect 9, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, and a light chain CDR of SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204.

27. The human antibody agent of aspect 10, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, and a light chain CDR of SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207.

28. The human antibody agent of aspect 11, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102, and a light chain CDR of SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO: 210.

29. The human antibody agent of aspect 12, wherein the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, and the light chain CDRs of SEQ ID NO: 211, SEQ ID NO: 212 and SEQ ID NO: 213.

30. The human antibody agent of aspect 13, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108, and a light chain CDR of SEQ ID NO: 214, SEQ ID NO: 215 and SEQ ID NO: 216.

31. The human antibody agent of aspect 14, wherein the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, and the light chain CDRs of SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219.

32. The human antibody agent of aspect 15, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, and a light chain CDR of SEQ ID NO: 220, SEQ ID NO: 221 and SEQ ID NO: 222.

33. The human antibody agent of aspect 16, wherein the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, and the light chain CDRs of SEQ ID NO: 223, SEQ ID NO: 224 and SEQ ID NO: 225.

34. The human antibody agent of aspect 17, wherein the human antibody agent comprises a heavy chain CDR of SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, and a light chain CDR of SEQ ID NO: 226, SEQ ID NO: 227 and SEQ ID NO: 228.

35. The human antibody agent of aspect 18, wherein the human antibody agent comprises the heavy chain CDRs of SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123, and the light chain CDRs of SEQ ID NO: 229, SEQ ID NO: 230 and SEQ ID NO: 231.

36. A human antibody agent that competes for CD19 binding with a human antibody agent that comprises (a) a heavy chain CDR of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, and a light chain CDR SEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183; (b) heavy chain CDRs of SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78 and light chain CDRs of SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186; (c) heavy chain CDRs of SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81 and light chain CDRs of SEQ ID NO: 187, SEQ ID NO: 188 and SEQ ID NO: 189; (d) heavy chain CDRs of SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84 and light chain CDRs of SEQ ID NO: 190, SEQ ID NO: 191 and SEQ ID NO: 192; (e) heavy chain CDRs of SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87 and light chain CDRs of SEQ ID NO: 193, SEQ ID NO: 194 and SEQ ID NO: 195; (f) heavy chain CDRs of SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90 and light chain CDRs of SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198; (g) heavy chain CDRs of SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, and light chain CDRs of SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201; (h) heavy chain CDRs of SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96 and light chain CDRs of SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204; (i) heavy chain CDRs of SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99 and light chain CDRs of SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207; (j) heavy chain CDRs of SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102 and light chain CDRs of SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO: 210; (k) heavy chain CDRs of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, and light chain CDRs of SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO: 213; (l) heavy chain CDRs of SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108 and light chain CDRs of SEQ ID NO: 214, SEQ ID NO: 215 and SEQ ID NO: 216; (m) heavy chain CDRs of SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111 and light chain CDRs of SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219; (n) heavy chain CDRs of SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, and light chain CDRs of SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO: 222; (o) heavy chain CDRs of SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, and light chain CDRs of SEQ ID NO: 223, SEQ ID NO: 224, and SEQ ID NO: 225; (p) heavy chain CDRs of SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120 and light chain CDRs of SEQ ID NO: 226, SEQ ID NO: 227 and SEQ ID NO: 228; or (q) heavy chain CDRs of SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, and light chain CDRs of SEQ ID NO: 229, SEQ ID NO: 230, and SEQ ID NO: 231.

37. The human antibody agent of any one of aspects 1-36, wherein the human antibody agent further comprises one or more amino acid substitutions in a heavy chain CDR and/or a light chain CDR.

38. The human antibody agent of aspect 37, wherein the human antibody agent further comprises at least one or up to five amino acid substitutions in a heavy chain CDR and/or a light chain CDR.

39. The human antibody agent of aspect 38, wherein the human antibody agent further comprises at least one or up to three amino acid substitutions in a heavy chain CDR and/or a light chain CDR.

40. A human antibody agent that specifically binds human CD19, wherein the human antibody agent comprises a heavy chain variable region has a sequence that is at least 95% identical to the heavy chain variable region sequence as shown in Table 1 , and the light chain variable region has a sequence that is at least 95% identical to the light chain variable region sequence as shown in Table 1.

41. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 2 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 2.

42. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 4 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 4.

43. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 6 and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 6.

44. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 8 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 8.

45. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 10 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 10.

46. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 12 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 12.

47. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 14 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 14.

48. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 16 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 16.

49. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 18 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 18.

50. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 20. , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 20.

51. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 22 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 22.

52. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 24 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 24.

53. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 26 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 26.

54. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 28 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 28.

55. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 30. , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 30.

56. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to a heavy chain variable region sequence as set forth in SEQ ID NO: 32 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 32.

57. The human antibody agent of aspect 40, wherein the human antibody agent comprises a heavy chain variable region having a sequence that is at least 95% identical to the heavy chain variable region sequence as set forth in SEQ ID NO: 34 , and a light chain variable region having a sequence that is at least 95% identical to the light chain variable region sequence as set forth in SEQ ID NO: 34.

58. The human antibody agent of any one of aspects 40-57, wherein the human antibody agent comprises the heavy and light chain variable region sequences as set forth in any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO : 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 , SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 34.

59. A human antibody agent that specifically binds human CD19, wherein the human antibody agent comprises one or more amino acid substitutions compared to the parent human antibody agent, and wherein the human antibody agent comprises (a) a heavy chain CDR of SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126, and light chain CDRs of SEQ ID NO: 232, SEQ ID NO: 233, and SEQ ID NO: 234; (b) heavy chain CDRs of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, and light chain CDRs of SEQ ID NO: 235, SEQ ID NO: 236, and SEQ ID NO: 237; (c) heavy chain CDRs of SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132 and light chain CDRs of SEQ ID NO: 238, SEQ ID NO: 239 and SEQ ID NO: 240; (d) heavy chain CDRs of SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135 and light chain CDRs of SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 243; (e) heavy chain CDRs of SEQ ID NO: 136, SEQ ID NO: 137 and SEQ ID NO: 138 and light chain CDRs of SEQ ID NO: 244, SEQ ID NO: 245 and SEQ ID NO: 246; (f) heavy chain CDRs of SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141 and light chain CDRs of SEQ ID NO: 247, SEQ ID NO: 248 and SEQ ID NO: 249; (g) heavy chain CDRs of SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144, and light chain CDRs of SEQ ID NO: 250, SEQ ID NO: 251, and SEQ ID NO: 252; (h) heavy chain CDRs of SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 147 and light chain CDRs of SEQ ID NO: 253, SEQ ID NO: 254 and SEQ ID NO: 255; (i) heavy chain CDRs of SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150, and light chain CDRs of SEQ ID NO: 256, SEQ ID NO: 257, and SEQ ID NO: 258; (j) heavy chain CDRs of SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153 and light chain CDRs of SEQ ID NO: 259, SEQ ID NO: 260 and SEQ ID NO: 261; (k) heavy chain CDRs of SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156 and light chain CDRs of SEQ ID NO: 262, SEQ ID NO: 263 and SEQ ID NO: 264; (l) heavy chain CDRs of SEQ ID NO: 157, SEQ ID NO: 158 and SEQ ID NO: 159 and light chain CDRs of SEQ ID NO: 265, SEQ ID NO: 266 and SEQ ID NO: 267; (m) heavy chain CDRs of SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 163 and light chain CDRs of SEQ ID NO: 268, SEQ ID NO: 269 and SEQ ID NO: 270; (n) heavy chain CDRs of SEQ ID NO: 163, SEQ ID NO: 164, and SEQ ID NO: 165, and light chain CDRs of SEQ ID NO: 271, SEQ ID NO: 272, and SEQ ID NO: 273; (o) heavy chain CDRs of SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, and light chain CDRs of SEQ ID NO: 274, SEQ ID NO: 275, and SEQ ID NO: 276; (p) heavy chain CDRs of SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 171 and light chain CDRs of SEQ ID NO: 277, SEQ ID NO: 278 and SEQ ID NO: 279; (q) heavy chain CDRs of SEQ ID NO: 172, SEQ ID NO: 173 and SEQ ID NO: 174 and light chain CDRs of SEQ ID NO: 280, SEQ ID NO: 281 and SEQ ID NO: 282; (r) heavy chain CDRs of SEQ ID NO: 175, SEQ ID NO: 176, and SEQ ID NO: 177, and light chain CDRs of SEQ ID NO: 283, SEQ ID NO: 284, and SEQ ID NO: 285; or (s) heavy chain CDRs of SEQ ID NO: 178, SEQ ID NO: 179, and SEQ ID NO: 180, and light chain CDRs of SEQ ID NO: 286, SEQ ID NO: 287, and SEQ ID NO: 288.

60. The human antibody agent of aspect 59, wherein the human antibody agent comprises a light chain variable region that includes one or more amino acid substitutions at any of the amino acid positions selected from the group consisting of 10, 16, 25, 34, 52, 54, 68, 69, 72, 75, 93, 95 and combinations thereof.

61. The human antibody agent of aspect 60, wherein the one or more amino acid substitutions include V10M, K16E, S25N, V34I, D52N, L54Q, N68T, T69M, L72M, S75N, S93T, D95E, D95G, or a combination thereof.

62. The human antibody agent of any one of aspects 59-61, wherein the human antibody agent includes a heavy chain variable region that includes one or more amino acid substitutions at any of the amino acid positions selected from the group consisting of 3, 12, 16, 17, 25, 26, 32, 63, 69, 97, 102, 106, 108, 109, 113, 116, 117 and combinations thereof.

63. The human antibody agent of aspect 62, wherein the one or more amino acid substitutions include Q3R, K12E, E16G, S17F, S25A, G26A, Y32F, S63F, T69A, A97V, T102S, M106L, Y108N, D109E, Q113L, L116M, M117L or combinations thereof.

64. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with the amino acid substitution S75N.

65. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with the amino acid substitution T69M.

66. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with amino acid substitutions D52N and D95E.

67. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with amino acid substitutions V10M and D95G.

68. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with amino acid substitution S93T and a heavy chain variable region with amino acid substitutions S17F and T69A.

69. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with amino acid substitution N68T and a heavy chain variable region with amino acid substitutions S17F and Y108N.

70. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a heavy chain variable region with the amino acid substitution S63F.

71. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a heavy chain variable region with the amino acid substitutions Q3R, Y32F, and A97V.

72. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with the amino acid substitution K16E.

73. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a heavy chain variable region with the amino acid substitution M117L.

74. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with amino acid substitution L72M and a heavy chain variable region with amino acid substitution S25A.

75. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a heavy chain variable region with amino acid substitutions T102S and L116M.

76. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with amino acid substitutions S25N and V34I and a heavy chain variable region with amino acid substitutions K12E and D109E.

77. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a heavy chain variable region with an E16G amino acid substitution.

78. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with amino acid substitution L54Q and a heavy chain variable region with amino acid substitution M106L.

79. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with the amino acid substitution S25N.

80. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a heavy chain variable region with amino acid substitutions G26A and Q113L.

81. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a light chain variable region with the amino acid substitution L54Q.

82. The human antibody agent of any one of aspects 59-63, wherein the human antibody agent comprises a heavy chain variable region with the amino acid substitution T102S.

83. The human antibody agent of any one of aspects 59-82, wherein the human antibody agent comprises the heavy and light chain variable region sequences as set forth in any of SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO : 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 , SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.

84. The human antibody agent of any one of aspects 1-83, wherein the human antibody agent is a human monoclonal antibody that includes a variant Fc region.

85. The human antibody agent of aspect 84, wherein the human monoclonal antibody includes a variant glycosylation pattern compared to the parental human monoclonal antibody that includes a wild-type Fc region.

86. The human antibody agent of any one of aspects 1-85, wherein the human antibody agent is conjugated to a therapeutic agent or detection agent.

87. The human antibody agent of any one of aspects 1-85, wherein the human antibody agent is conjugated to a cytotoxic agent or group.

88. The human antibody agent of any one of aspects 1-85, wherein the human antibody agent is conjugated to a radioisotope.

89. The human antibody agent of any one of aspects 1-88, wherein the human antibody agent is or includes an immunoglobulin, Fab, F(ab') ₂ , Fd, Fv, single chain Fv (scFv), or dAb.

90. The human antibody agent of aspect 89, wherein the human antibody agent is or includes scFv.

91. The human antibody agent of aspect 90, wherein the scFv includes a linker sequence.

92. The human antibody agent of aspect 90 or 91, wherein the scFv is conjugated to a therapeutic agent or detection agent.

93. The human antibody agent of aspect 90 or 91, wherein the scFv is part of a chimeric antigen receptor.

94. The human antibody agent of any one of aspects 1-93 for use in therapy or diagnosis.

95. The human antibody agent of any one of aspects 1-93 for use in the treatment, prevention, or amelioration of a disease characterized by CD19 expression.

96. The use of a human antibody agent according to any one of aspects 1-93 for use in the treatment, prevention, or alleviation of cancer.

97. An isolated nucleic acid molecule encoding a human antibody agent according to any one of aspects 1-93.

98. The isolated nucleic acid molecule of aspect 97, wherein the isolated nucleic acid molecule includes a codon optimized sequence.

99. The isolated nucleic acid molecule of aspect 97, wherein the isolated nucleic acid molecule is a sequence that is shown in any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69 and SEQ ID NO: 71.

100. A vector comprising an isolated nucleic acid molecule according to any one of aspects 97-99.

101. A cell containing a vector according to aspect 100.

102. A kit comprising a human antibody agent according to any one of aspects 1-93, an isolated nucleic acid molecule according to any one of aspects 97-99, a vector according to aspect 100, or a cell according to aspect 101.

103. A vaccine comprising the isolated nucleic acid molecule according to any one of aspects 97-99.

104. A method for producing a human antibody agent according to any one of aspects 1-93, comprising the step of culturing the cell according to aspect 101 in a culture medium under conditions allowing expression of the human antibody agent and separating the human antibody agent from the culture medium.

105. A composition comprising a human antibody agent according to any one of aspects 1-93.

106. The composition of aspect 105 wherein the human antibody agent is conjugated to a cytotoxic agent or fragment.

107. A pharmaceutical composition comprising the composition of option 105 or 106 or the human antibody agent of any one of aspects 1-93 and further comprising a pharmaceutically acceptable carrier or diluent.

108. A method of treating a medical condition characterized by CD19 expression in a subject, comprising the step of administering a therapeutically effective amount of a human antibody agent according to any one of aspects 1-93 to the subject.

109. The method of aspect 108, wherein the medical condition characterized by CD19 expression is B-cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt's lymphoma, non-Hodgkin's lymphoma, or acute myeloid leukemia.

110. The method of aspect 108, wherein the medical condition characterized by CD19 expression is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), diabetes, or scleroderma.

111. A method of treating cancer, comprising the step of administering to a subject a human antibody agent according to any one of aspects 1-93.

112. The use of an antibody agent according to any one of aspects 1-93 for treating or detecting a condition associated with CD19 expression.

113. A bispecific antibody comprising a first antigen-binding site from a human antibody agent according to any one of aspects 1-93 and a second antigen-binding site.

114. The bispecific antibody of aspect 113, wherein the first and second antigen binding sites are scFv.

115. The bispecific antibody of aspect 113, wherein the first antigen binding site consists of an immunoglobulin molecule and the second antigen binding site consists of scFv, scFab, Fab, or Fv.

116. The bispecific antibody of any one of aspects 113-115, wherein the second antigen binding site binds an immune cell selected from the group consisting of T cells, NK cells, B cells, dendritic cells, monocytes, macrophages, neutrophils, mesenchymal stem cells and neural stem cells.

117. The bispecific antibody of aspect 116, wherein the second antigen binding site binds CD3 on T cells.

118. A bispecific T cell monoclonal antibody comprising the antigen binding site of the human antibody agent of any one of aspects 1-93.

119. A bispecific antibody according to any one of aspects 113-118 for use in therapy or diagnostics.

120. An isolated nucleic acid molecule that encodes in whole or in part the polypeptide chain of a bispecific antibody according to any one of aspects 113-118.

121. The isolated nucleic acid molecule of aspect 120, wherein the nucleic acid molecule includes a codon optimized sequence.

122. A vector comprising a nucleic acid sequence according to aspect 120 or 121.

123. Cell containing the vector according to aspect 122.

124. A kit comprising a bispecific antibody according to any one of aspects 113-118, isolated nucleotide molecules according to aspect 120 or 121, a vector according to aspect 122, or a cell according to aspect 123.

125. A composition comprising a bispecific antibody according to any one of aspects 113-118.

126. A pharmaceutical composition comprising the composition of aspect 125 or the bispecific antibody of any of aspects 113-118 and further comprising a pharmaceutically acceptable carrier or diluent.

127. A chimeric antigen receptor comprising an antigen-binding site of a human antibody agent according to any one of aspects 1-93.

128. The chimeric antigen receptor of aspect 127, wherein the antigen binding site is or includes scFv.

129. The chimeric antigen receptor of aspect 127, wherein the antigen binding site is a V _L region.

130. The chimeric antigen receptor of aspect 129, wherein the V _L region comprises a light chain CDR of (a) SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO: 183; (b) SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186; (c) SEQ ID NO: 187, SEQ ID NO: 188 and SEQ ID NO: 189; (d) SEQ ID NO: 190, SEQ ID NO: 191 and SEQ ID NO: 192; (e) SEQ ID NO: 193, SEQ ID NO: 194 and SEQ ID NO: 195; (f) SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198; (g) SEQ ID NO: 199, SEQ ID NO: 200 and SEQ ID NO 201; (h) SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204; (i) SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207;

(j) SEQ ID NO:208, SEQ ID NO:209 and SEQ ID NO:210; (k) SEQ ID NO:211, SEQ ID NO:212 and SEQ ID NO:213; (l) SEQ ID NO:214, SEQ ID NO:215 and SEQ ID NO:216;

(m) SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219; (n) SEQ ID NO: 220, SEQ ID NO: 221 and SEQ ID NO: 222; (o) SEQ ID NO: 223, SEQ ID NO: 224 and SEQ ID NO: 225; (p) SEQ ID NO: 226, SEQ ID NO: 227 and SEQ ID NO: 228; (q) SEQ ID NO: 229, SEQ ID NO: 230 and SEQ ID NO: 231; (r) SEQ ID NO: 232, SEQ ID NO: 233 and SEQ ID NO: 234; (s) SEQ ID NO: 235, SEQ ID NO: 236 and SEQ ID NO: 237; (t) SEQ ID NO: 238, SEQ ID NO: 239 and SEQ ID NO: 240; (u) SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 243; (v) SEQ ID NO: 244, SEQ ID NO: 245 and SEQ ID NO: 246; (w) SEQ ID NO: 247, SEQ ID NO: 248 and SEQ ID NO: 249; (x) SEQ ID NO: 250, SEQ ID NO: 251 and SEQ ID NO: 252; (y) SEQ ID NO: 253, SEQ ID NO: 254 and SEQ ID NO: 255; (z) SEQ ID NO: 256, SEQ ID NO: 257 and SEQ ID NO: 258; (ab) SEQ ID NO: 259, SEQ ID NO: 260 and SEQ ID NO: 261; (AC) SEQ ID NO: 262, SEQ ID NO: 263 and SEQ ID NO: 264; (ad) SEQ ID NO: 265, SEQ ID NO: 266 and SEQ ID NO: 267; (ae) SEQ ID NO: 268, SEQ ID NO: 269 and SEQ ID NO: 270; (af) SEQ ID NO: 271, SEQ ID NO: 272 and SEQ ID NO: 273; (ag) SEQ ID NO: 274, SEQ ID NO: 275 and SEQ ID NO: 276; (a) SEQ ID NO: 277, SEQ ID NO: 278 and SEQ ID NO: 279; (ai) SEQ ID NO: 280, SEQ ID NO: 281 and SEQ ID NO: 282; (aj) SEQ ID NO: 283, SEQ ID NO: 284 and SEQ ID NO: 285; or (ak) SEQ ID NO: 286, SEQ ID NO: 287 and SEQ ID NO: 288.

131. The chimeric antigen receptor of aspect 129, wherein the V _L region comprises a sequence as shown in any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO : 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 , SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.

132. The chimeric antigen receptor of aspect 127, wherein the antigen binding site is a V _H region.

133. The chimeric antigen receptor of aspect 132, wherein the V _H region comprises the heavy chain CDRs of (a) SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75; (b) SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78; (c) SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81; (d) SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84; (e) SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87; (f) SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90; (g) SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93; (h) SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96; (i) SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99; (j) SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102; (k) SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105; (1) SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108; (m) SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111; (n) SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114; (o) SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117; (p) SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120; (q) SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123; (r) SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126; (s) SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129; (t) SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132; (u) SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135; (v) SEQ ID NO: 136, SEQ ID NO: 137 and SEQ ID NO: 138; (w) SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141; (x) SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144; (y) SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 147; (z) SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150; (ab) SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153; (AC) SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156; (ad) SEQ ID NO: 157, SEQ ID NO: 158 and SEQ ID NO: 159; (ae) SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 163; (af) SEQ ID NO: 163, SEQ ID NO: 164 and SEQ ID NO: 165; (ag) SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168; (a) SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 171; (ai) SEQ ID NO: 172, SEQ ID NO: 173 and SEQ ID NO: 174; (aj) SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177; or (ak) SEQ ID NO: 178, SEQ ID NO: 179 and SEQ ID NO: 180.

134. The chimeric antigen receptor of aspect 132, wherein the V _H region comprises a sequence as shown in any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO : 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 , SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.

135. A chimeric antigen receptor according to any one of aspects 127-134 for use in therapy or diagnostics.

136. A chimeric antigen receptor according to any one of aspects 127-134 for use in the treatment, prevention, or alleviation of a disease characterized by CD19 expression.

137. An isolated nucleic acid molecule that encodes a chimeric antigen receptor according to any one of aspects 127-134.

138. The isolated nucleic acid molecule of aspect 137, wherein the nucleic acid molecule includes a codon optimized sequence.

139. An effector immune cell that expresses a chimeric antigen receptor according to any one of aspects 127-134.

140. The effector immune cell of aspect 139, wherein the cell is a T cell or a NK cell.

141. An effector immune cell in aspect 139 or 140 for use in therapy or diagnosis.

142. An effector immune cell according to aspect 139 or 140 for use in the treatment, prevention, or alleviation of a disease characterized by CD19 expression.

143. A kit comprising a chimeric antigen receptor according to any one of aspects 127-134, an isolated nucleic acid molecule according to aspect 137 or 138, or an immune effector cell according to aspect 139 or 140.

144. The use of a chimeric antigen receptor according to any one of aspects 127-134 for the treatment or detection of a medical condition associated with CD19 expression.

145. A method for inhibiting tumor growth, comprising the step of contacting tumor cells with a bispecific antibody, wherein the bispecific antibody consists of a first antigen-binding site based on an agent based on a human antibody according to any one of aspects 1-93 and a second antigen-binding site that binds an immune cell, wherein the contact carried out under conditions and for a time sufficient to observe the destruction of tumor cells.

146. A method for killing tumor cells, comprising the step of contacting tumor cells with a bispecific antibody, wherein the bispecific antibody consists of a first antigen-binding site based on an agent based on a human antibody according to any one of aspects 1-93 and a second antigen-binding site that binds an immune cell, wherein the contact carried out under conditions and for a time sufficient to inhibit the growth of tumor cells.

147. The method of aspect 145 or 146 wherein the immune cell is a T cell or NK cell.

148. The method of any one of aspects 145-147, wherein the first and second antigen binding sites are scFv.

149. The method of aspect 148 wherein the second antigen binding site binds CD3 on T cells.

150. A bispecific antibody consisting of a first antigen-binding site based on a human antibody agent according to any one of aspects 1-93 and a second antigen-binding site that binds an immune cell, for use in inhibiting tumor growth.

151. A bispecific antibody consisting of a first antigen-binding site based on a human antibody agent according to any one of aspects 1-93 and a second antigen-binding site that binds an immune cell, for use in killing tumor cells.

152. A method of directing T cells to kill target cells expressing CD19, the method comprising the step of contacting one or more target cells expressing CD19 with a bispecific antibody, the bispecific antibody comprising a first antigen-binding site of an agent based on a human antibody according to any one of of aspects 1-93 and a second antigen-binding site that binds CD3 on T cells, wherein the contact is made under conditions and for a time sufficient for the T cells to which the bispecific antibody binds to mediate the killing of target cells expressing CD19 .

153. The method of aspect 152, wherein the first and second antigen binding sites are scFv.

154. A bispecific antibody consisting of a first antigen-binding site based on a human antibody agent according to any one of aspects 1-93 and a second antigen-binding site that binds CD3 on T cells, for use in targeting T cells to kill target cells, expressing CD19.

155. A bispecific antibody consisting of a first scFv that specifically binds CD19 and a second scFv that specifically binds CD3 on T cells, where the N-terminus of the first scFv is linked to the C-terminus of the second scFv and where the first scFv includes any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.

156. The bispecific antibody of aspect 155, wherein the N-terminus of the first scFv is linked to the C-terminus of the second scFv via a linker sequence.

157. The bispecific antibody of aspect 156, wherein the linker sequence includes a (G ₄ S) _n linker.

158. The bispecific antibody of aspect 157, wherein n is 1, 2, 3, 4, or 5.

159. The bispecific antibody of aspect 156, wherein the linker sequence is SRGGGGGGGGGGGGGGGEMEMA (SEQ ID NO: 289).

160. A pharmaceutical composition comprising the bispecific antibody of any one of aspects 155-159 and further comprising a pharmaceutically acceptable carrier or diluent.

161. A bispecific antibody according to any one of aspects 155-159 for use in therapy or diagnostics.

162. An antibody-drug conjugate comprising an antigen-binding site of a human antibody agent according to any one of aspects 1-93 and a cytotoxic agent or group.

163. A kit for diagnosing an object suffering from a disease associated with CD19 or a predisposition to it, or for predicting the state of the object, where the kit includes a detecting tool for detecting the concentration of CD19 present in a sample from the test object, where the detecting tool includes an antigen-binding site a human antibody agent according to any one of aspects 1-93, a bispecific binding agent according to any one of aspects 113-118 and 155-159, a chimeric antigen receptor according to any one of aspects 127-134, or an immune effector cell according to any aspect 138 or 139, each of which is optionally derivatized, wherein the presence of CD19 in the sample indicates that the subject is suffering from a disease associated with CD19.

Equivalents

Application of ordinal terms such as "first", "second", "third", etc. in the claims for modifying an item element, does not by itself imply any priority, preference, or order of one item item over another, or the temporal order in which the steps of the method are performed, but are used simply as labels to distinguish one method element having a specific name from another. an element having the same name (but to apply the ordinal term) in order to distinguish between the elements of the clause.

It should be understood that the words in the singular used in this document in the description and in the claims, unless expressly indicated otherwise, include the plural. Claims or descriptions that include "or" between one or more members of a group are deemed to be satisfied if one, more than one, or all of the members of the group are present, applied, or related to a particular product or process, unless otherwise stated or otherwise evident from context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. The invention also includes embodiments in which more than one or all of the members of the group are present, applied, or related to a particular product or method. In addition, it should be understood that the invention covers all variants, combinations and permutations in which one or more restrictions, elements, provisions, descriptive terms, etc. of one or more of the listed clauses are introduced into another clause dependent on the same base clause (or, if applicable, any other clause), unless otherwise noted, or unless it is obvious to a person skilled in the art that a contradiction or inconsistency would arise. Where elements are presented as lists (eg, in a Markush group or similar format), it should be understood that each subgroup of elements is also disclosed and any element(s) may be removed from the group. It should be understood that, in general, when the invention or aspects of the invention are referred to as containing specific elements, features, etc., some embodiments of the invention or aspects of the invention consist or consist essentially of such elements, features, etc. For simplicity, these embodiments have not been specifically set forth in so many words in this document on a case-by-case basis. It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exception is indicated in the description. Publications, websites and other reference materials mentioned in this document to describe the background of the invention and provide additional details regarding its practical implementation, are incorporated herein by reference.

Thus, having described several aspects of at least one embodiment of the present invention, it should be understood that various changes, modifications, and improvements will be apparent to those skilled in the art. Such changes, modifications and improvements are intended to be disclosed in part and are intended to be within the spirit and scope of the invention. Accordingly, the above description and drawings are by way of example only, and the invention is described in detail in the claims below.

--->

SEQUENCE LISTING

<110> Eureka Therapeutics Inc.

<120> ANTIBODY AGENTS SPECIFIC FOR HUMAN CD19 AND USES THEREOF

<130> ET-2015-11-WO-PCT

<140> US 62/240,624

<141> 2015-10-13

<160> 302

<170>PatentIn version 3.5

<210> 1

<211> 747

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<213> Artificial Sequence

<220>

<223> Synthetic Construct

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cagactgtgg tgactcagga gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60

tcctgctctg gaagcagctc caacattggg aataattatg tatcctggta ccagcagctc 120

cggaacag cccccaaact cctcatttat gacaataata agcgaccctc agggattcct 180

gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240

actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tgctggtgtc 300

ttcggaactg ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctggt ggagactgggg 420

ggaggcttgg tacagcctgg ggggtccctg agactctcct gtgcagcctc tggattcacc 480

tttagcagct atgccatgag ctgggtccgc caggctccag ggaaggggct ggagtgggtc 540

tcagctatta gtggtagtgg tggtagcaca tactacgcag actccgtgaa gggccggttc 600

accatctcca gagacaattc caagaacacg ctgtatctgc aaatgaacag cctgagagcc 660

gaggacacgg ccgtatatta ctgtgcgcgc tactactact ctcgtctggga ttactggggt 720

caaggtactc tggtgaccgt ctcctca 747

<210> 2

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 2

Gln Thr Val Val Thr Gln Glu Pro Ser Val Ser Ala Ala Pro Gly Gln

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Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

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Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

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Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

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Ser Ala Gly Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val

130 135 140

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

145 150 155 160

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr

180 185 190

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

195 200 205

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Ala Arg Tyr Tyr Tyr Ser Arg Leu Asp Tyr Trp Gly

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Gln Gly Thr Leu Val Thr Val Ser Ser

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cagtctgtgc tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60

tcctgctctg gaagcagctc caacattggg aataattatg tatcctggta ccggcaactc 120

cggaacag cccccaaact cctcatctat gaaaataata agcgaccctc agggattcct 180

gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240

actggggacg aggccgatta ttactgcgga acatgggata gcagcctgcg tgctggggtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctggt ggagtctgggg 420

ggaggcttgg tacagcctgg ggggtccctg agactctcct gtgcagcctc tggattcacc 480

tttagcagct atgccatgag ctgggtccgc caggctccgg ggaaggggct ggagtgggtc 540

tcaggtatta gtgctagtgg tggtagcaca tactacgcag actccgtgaa gggccgcttc 600

accatctcca gagacaattc caagaatacg ctgtatctgc aaatgaacag cctgagagcc 660

gaggacacgg ccgtatatta ctgtgcgcgc tactacctgt ctcagatcga ttcttggggt 720

caaggtactc tggtgaccgt ctcctca 747

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<213> Artificial Sequence

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Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Gly Gln

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Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

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Tyr Val Ser Trp Tyr Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

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Ile Tyr Glu Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Arg Ala Gly Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Leu Glu Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

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Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

145 150 155 160

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

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Leu Glu Trp Val Ser Gly Ile Ser Ala Ser Gly Gly Ser Thr Tyr Tyr

180 185 190

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

195 200 205

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Ala Arg Tyr Tyr Leu Ser Gln Ile Asp Ser Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser

245

<210> 5

<211> 753

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<213> Artificial Sequence

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<223> Synthetic Construct

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cagtctgtgt tgactcagcc accctcagcg tctgggaccc ccggggcagag ggtcaccatc 60

120

cgggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggccatgtg 300

gtattcggcg gagggaccaa gctgaccgtc ctaggttcta gaggtggtgg tggtagcggc 360

ggcggcggct ctggtggtgg tggatccctc gagatggccg aggtccagct ggtgcagtct 420

ggggctgagg tgaagaagcc tggggctaca gtgaaaatct cctgcaaggt ttctggatac 480

accttcaccg actactacat gcactgggtg caacaggccc ctggaaaagg gcttgagtgg 540

atgggacttg ttgatcctga agatggtgaa acaatatacg cagagaagtt ccagggcaga 600

gtcaccataa ccgcggacac gtctacagac acagcctaca tggagctgag cagcctgaga 660

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<210> 6

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

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Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

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Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

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Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

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Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

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Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

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Asn Gly His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

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Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

130 135 140

Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr

145 150 155 160

Thr Phe Thr Asp Tyr Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys

165 170 175

Gly Leu Glu Trp Met Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile

180 185 190

Tyr Ala Glu Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser

195 200 205

Thr Asp Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

210 215 220

Ala Val Tyr Tyr Cys Ala Thr Gly Ile Tyr Ser Arg Pro Leu Gly Tyr

225 230 235 240

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 7

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 7

tcctatgtgc tgactcagcc accctcagcg tctgggaccc ccggggcagag ggtcaccatc 60

tcttgttctg gaagcagctc caacatcgga agtcatactg taaactggta ccagcagctc 120

cgggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgagg tgcagctggt ggagactggg 420

ggaggcttgg tacagcctgg ggggtccctg agactctcct gtgcagcctc tggattcacc 480

tttagcagct atgccatgag ctgggtccgc caggctccag ggaaggggct ggagtgggtc 540

tcagctatta gtggtagtgg tggtagcaca tactacgcag actccgtgaa gggccgattc 600

accatctcca gagacaattc caagaacacg ctgtatcttc aaatgaacag cctgagagct 660

gaggacacgg ccgtgtatta ctgtgcgcgc tctgacggta aacatttctg gcagcagtac 720

gatgcttggg gtcaaggtac tctggtgacc gtctcctca 759

<210> 8

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 8

Ser Tyr Val Leu Thr Gln Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser His

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val

130 135 140

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

145 150 155 160

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr

180 185 190

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

195 200 205

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Ala Arg Ser Asp Gly Lys His Phe Trp Gln Gln Tyr

225 230 235 240

Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 9

<211> 729

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 9

gacatccagt tgacccagtc tccatcctcc ctgtctgcat atgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca gggcattacc aattctttag cctggtatca gcagaagcca 120

gggaaagccc ctaagctcct gctacatgct gcatccagat tggagtctgg ggtcccatcc 180

aggttcagtg gcagtggatt tgggacggat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg cagtttatta ctgtcaacac tatttaggta ccccgtactc ttttggccag 300

gggaccaagg tggagatcaa acgttctaga ggtggtggtg gtagcggcgg cggcggctct 360

ggtggtggtg gatccctcga gatggccgag gtgcagctgg tggagtctgg aggaggcttg 420

gtccagcctg gggggtccct gagactctcc tgtgcagcct ctgggttcac cgtcagtagc 480

aactacatga gctgggtccg ccaggctcca gggaaggggc tggagtgggt ctcagctatt 540

agtggtagtg gtggtagcac atactacgca gactccgtga agggccggtt caccatctcc 600

agagacaatt ccaagaacac gctgtatctg caaatgaaca gcctgagagc cgaggacacg 660

gccgtatatt actgtgcgcg catgaacatc gattactggg gtcaaggtac tctggtgacc 720

gtctcctca 729

<210> 10

<211> 243

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 10

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Tyr Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Thr Asn Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

His Ala Ala Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Leu Gly Thr Pro Tyr

85 90 95

Ser Phe Gly Glyn Gly Thr Lys Val Glu Ile Lys Arg Ser Arg Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Met

115 120 125

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser

145 150 155 160

Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Met Asn Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser

<210> 11

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 11

gaaattgtgc tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atctcttgcc gggcaagtca gagcgttagc agatttttaa attggtatca gcagaaaccg 120

ggtaaagccc ctaagctcct gatctatggt gtatccactt tggaacgtgg ggtcccttca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaagag agttacatta tcccgctcac tttcggcgga 300

gggaccaagc tggagatcaa acgttctaga ggtggtggtg gtagcggcgg cggcggctct 360

ggtggtggtg gatccctcga gatggccgag gtgcagctgg tgcagtctgg agcagaggtg 420

aaaaggcccg gggagtctct gacgatctcc tgtaagggtt ctgaatacag ctttgccagc 480

tactggatca cctgggtgcg ccagatgccc gggaaaggcc tggagtggat ggggaggatt 540

gatcctagtg actcttatac caactacagc ccgtccttcc aaggccacgt caccatctca 600

gctgacaagt ccatcagcac tgcctacttg cagtggagca gcctgaaggc ctcggacacc 660

gccatatatt actgtgcgag accttttcag tacgactacg gtggttactc cgatgctttt 720

gatatctggg gccaagggac aatggtcacc gtctcttca 759

<210> 12

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 12

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Val Ser Thr Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Ser Tyr Ile Ile Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ser Arg Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Met

115 120 125

Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly

130 135 140

Glu Ser Leu Thr Ile Ser Cys Lys Gly Ser Glu Tyr Ser Phe Ala Ser

145 150 155 160

Tyr Trp Ile Thr Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

165 170 175

Met Gly Arg Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Ser Pro Ser

180 185 190

Phe Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

195 200 205

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr

210 215 220

Cys Ala Arg Pro Phe Gln Tyr Asp Tyr Gly Gly Tyr Ser Asp Ala Phe

225 230 235 240

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

245 250

<210> 13

<211> 753

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 13

gaaattgtga tgacgcagtc tccactctcc ctgtccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcttg gacagcaatg gattcaactc tttggattgg 120

tacctgcaga agccagggca gtctccacaa ctcctgatcc atttaggttc tgatcgggcc 180

tccggggtcc ctgacaggtt cagcggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggaatt tattactgca tgcagtctct acaaattccg 300

acgttcggcc aagggaccaa ggtggaaatc aaacgttcta gaggtggtgg tggtagcggc 360

ggcggcggct ctggtggtgg tggatccctc gagatggccc agatgcagct ggtgcaatct 420

ggggctgagg tgaagaaggc tgggtcctcg gtgaaggtct cctgcgagac ttctggaggc 480

accttcagca gctctagtgt caactgggtg cgacaggccc ctggacaagg gcttgagtgg 540

atgggaggaa tcatccctat cgttggaaca ccaaactacg cacagaagtt ccaggacaga 600

gtcacgatta ccgcggtcga atccaccttt acagcctaca tggagctgag cggcctgaga 660

tctgaggaca cggccgttta ttactgtgcg cgggggggat atcgcgacta tatggatgtc 720

tggggcagag ggaccacggt caccgtctcc tca 753

<210> 14

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 14

Glu Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asn Gly Phe Asn Ser Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile His Leu Gly Ser Asp Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Gln Ile Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Leu Glu Met Ala Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val

130 135 140

Lys Lys Ala Gly Ser Ser Val Lys Val Ser Cys Glu Thr Ser Gly Gly

145 150 155 160

Thr Phe Ser Ser Ser Ser Val Asn Trp Val Arg Gln Ala Pro Gly Gln

165 170 175

Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Val Gly Thr Pro Asn

180 185 190

Tyr Ala Gln Lys Phe Gln Asp Arg Val Thr Ile Thr Ala Val Glu Ser

195 200 205

Thr Phe Thr Ala Tyr Met Glu Leu Ser Gly Leu Arg Ser Glu Asp Thr

210 215 220

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Arg Asp Tyr Met Asp Val

225 230 235 240

Trp Gly Arg Gly Thr Thr Val Thr Val Ser Ser

245 250

<210> 15

<211> 747

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 15

tcctatgagc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60

120

cgggaacgg cccccaaact cctcatctat aggaataatc agcggccctc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag tggttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgagg tgcagctggt ggagtctgga 420

ggaggcttga tccagcctgg ggggtccctg agactctcct gtgcagcctc tgggttcacc 480

gtcagtagca actacatgag ctgggtccgc caggctccag ggaaggggct ggagtgggtc 540

tcagttattt atagcggtgg tagcacatac tacgcagact ccgtgaaggg ccgattcacc 600

atctccagag acaattccaa gaacacgctg tatcttcaaa tgaacagcct gagagccgag 660

gacacggccg tgtattactg tgcgagggggg gggtttggag ctgaatttga ctactggggc 720

cagggaaccc tggtcaccgt ctcctca 747

<210> 16

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 16

Ser Tyr Glu Leu Thr Gln Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile

130 135 140

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

145 150 155 160

Val Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala

180 185 190

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

195 200 205

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

210 215 220

Tyr Tyr Cys Ala Arg Gly Gly Phe Gly Ala Glu Phe Asp Tyr Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser

245

<210> 17

<211> 756

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 17

tcctatgtgc tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagataaatt gggggataaa tatgcttcct ggtatcagca gaagccaggc 120

cagtcccctg tactggtcat ctatcaagat aacaagcggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg actattactg tcaggcgtgg gacagcagca ctgaggatgt cttcggacct 300

gggaccaagg tcaccgtcct aggttctaga ggtggtggtg gtagcggcgg cggcggctct 360

ggtggtggtg gatccctcga gatggccgag gtgcagctgg tggagtctgg aggaggcttg 420

atccagcctg gggggtccct gagactctcc tgtgcagcct ctgggttcac cgtcagtagc 480

aactacatga gctgggtccg ccaggctcca gggaaggggc tggagtggggt ctcagttatt 540

tatagcggtg gtagcacata ctacgcagac tccgtgaagg gccgattcac catctccaga 600

gacaattcca agaacacgct gtatcttcaa atgaacagcc tgagagccga ggacacggcc 660

gtgtattact gtgcgagagg tggtatttcg gacgattact atggttcggg gagttatgat 720

aactggggcc agggaaccct ggtcaccgtc tcctca 756

<210> 18

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 18

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Glu Asp

85 90 95

Val Phe Gly Pro Gly Thr Lys Val Thr Val Leu Gly Ser Arg Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Met

115 120 125

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly

130 135 140

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser

145 150 155 160

Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

180 185 190

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

195 200 205

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Gly Gly Ile Ser Asp Asp Tyr Tyr Gly Ser Gly Ser Tyr Asp

225 230 235 240

Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 19

<211> 741

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 19

gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccccttcac tttcggcgga 300

gggaccaagg tggagatcaa acgttctaga ggtggtggtg gtagcggcgg cggcggctct 360

ggtggtggtg gatccctcga gatggccgag gtgcagctgg tggagtctgg gggaggcttg 420

gtccagcctg gggggtccct gagactctcc tgtgcagcct ctggattcac cgtcagtagc 480

aactacatga gctgggtccg ccaggctcca gggaaggggc tggagtggggt ctcagttatt 540

tatagcggtg gtagcacata ctacgcagac tccgtgaagg gccgattcac catctccaga 600

gacaattcca agaacacgct gtatcttcaa atgaacagcc tgagagctga ggacacggct 660

gtgtattact gtgcgagaga aagggggatg ggatatgctt ttgatatctg gggccaaggg 720

acaatggtca ccgtctcttc a 741

<210> 20

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 20

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Ser Arg Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Met

115 120 125

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser

145 150 155 160

Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

165 170 175

Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

180 185 190

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

195 200 205

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Glu Arg Gly Met Gly Tyr Ala Phe Asp Ile Trp Gly Gln Gly

225 230 235 240

Thr Met Val Thr Val Ser Ser

245

<210> 21

<211> 720

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 21

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gggcattggc agttatttag cctggtatca gcaaaaacca 120

gggaaagccc ctaaactcct gatctatcct gcatccacgt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacaa cttaatagtc tcttcggcca agggacacga 300

ctggagatta aacgttctag aggtggtggt ggtagcggcg gcggcggctc tggtggtggt 360

ggatccctcg agatggccca gctgcagctg caggagtcgg gcccaggact ggtgaagcct 420

tcggagaccc tgtccctcac ctgctctgtc tctggtgtct ccatgagtga aaactactgg 480

agctggatcc ggcagccccc agggaagcga ctggagtgga ttgggtgtgc ccattacact 540

ggggacaccc actacaaccc ctccctcaag ggtcgagtca ccatatcact agacacgtcc 600

atgaaccagt tctccctgag gctgaactct gtgaccgctg cggacacggc cgtctattac 660

tgtgcgagtt atcatccctt taactactgg ggccagggaa ccctggtcac cgtctcctca 720

<210> 22

<211> 240

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Pro Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Leu Phe Gly

85 90 95

Gln Gly Thr Arg Leu Glu Ile Lys Arg Ser Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Met Ala Gln Leu

115 120 125

Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu

130 135 140

Ser Leu Thr Cys Ser Val Ser Gly Val Ser Met Ser Glu Asn Tyr Trp

145 150 155 160

Ser Trp Ile Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile Gly Cys

165 170 175

Ala His Tyr Thr Gly Asp Thr His Tyr Asn Pro Ser Leu Lys Gly Arg

180 185 190

Val Thr Ile Ser Leu Asp Thr Ser Met Asn Gln Phe Ser Leu Arg Leu

195 200 205

Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Ser Tyr

210 215 220

His Pro Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

225 230 235 240

<210> 23

<211> 774

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 23

caggctgtgc tgactcagcc accctcggcg tctgggaccc ccggggcagag ggtcaccatc 60

tcttgttctg gaagcagctc caacatcgga actaaaactg taaactggta tcaggtgctc 120

cgggaacgg cccccaaact cctcatctat agtaattatc gccggccctc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgacgatg aggctgatta ttactgtgca ctatgggatg acagcctgga tggttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgagg tccagctggt gcagtctgggg 420

gctgaggtga ggaggcctgg ggctacagtg aaaatctcct gcaaggtttc tggatacacc 480

ttcaacgact tctacttaca ctgggtgcga caggcccctg gaaaagggct tgagtggatg 540

ggacgtattg atcctgaaga tggtaaaaca agatatgcag agaagttcca gggcagactc 600

accattaccg cggacacgtc tacagacact ctttacatgc aactgggcgg cctgacatct 660

gacgacacgg ccgtctatta ctgtacaact gattggggct atagcagttc cctacgtgag 720

gaggacatct ggtacgactg ctggggccag ggaaccctgg tcaccgtctc ctca 774

<210> 24

<211> 258

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 24

Gln Ala Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Lys

20 25 30

Thr Val Asn Trp Tyr Gln Val Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Tyr Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Asp Asp Ser Leu

85 90 95

Asp Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg

130 135 140

Arg Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr

145 150 155 160

Phe Asn Asp Phe Tyr Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Arg Ile Asp Pro Glu Asp Gly Lys Thr Arg Tyr

180 185 190

Ala Glu Lys Phe Gln Gly Arg Leu Thr Ile Thr Ala Asp Thr Ser Thr

195 200 205

Asp Thr Leu Tyr Met Gln Leu Gly Gly Leu Thr Ser Asp Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Thr Thr Asp Trp Gly Tyr Ser Ser Ser Leu Arg Glu

225 230 235 240

Glu Asp Ile Trp Tyr Asp Cys Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Ser

<210> 25

<211> 756

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 25

tcctatgagc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcat ctattatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatta tgtcttcgga 300

actgggacca aggtcaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaggtgcagc tggtgcagtc tggggctgag 420

gtgaagaagc ctgggtcctc ggtgaaggtc tcctgcaagg cttctggagg caccttcagc 480

agctatgcta tcagctgggt gcgacaggcc cctggacaag ggcttgagtg gatggggaggg 540

atcatcccta tctttggtac agcaaactac gcacagaagt tccagggcag agtcacgatt 600

accgcggacg aatccacgag cacagcctac atggagctga gcagcctgag atctgaggac 660

acggccgtgt attactgtgc gagagattat gggtacggtg actacggtga tgcttttgat 720

atctggggcc aagggacaat ggtcaccgtc tcttca 756

<210> 26

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 26

Ser Tyr Glu Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser

145 150 155 160

Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

165 170 175

Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln

180 185 190

Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr

195 200 205

Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

210 215 220

Tyr Cys Ala Arg Asp Tyr Gly Tyr Gly Asp Tyr Gly Asp Ala Phe Asp

225 230 235 240

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

245 250

<210> 27

<211> 753

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 27

tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaggccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg aaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattcctg tcaggtgtgg gatagcagta gtgatcatta tgtcttcgga 300

cctgggacca aggtcaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgcgttgtt ggtactatct actctatgca gtacgatgtt 720

tggggtcaag gtactctggt gaccgtctcc tca 753

<210> 28

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 28

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Ser Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Tyr Val Phe Gly Pro Gly Thr Lys Val Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Val Val Gly Thr Ile Tyr Ser Met Gln Tyr Asp Val

225 230 235 240

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 29

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 29

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 30

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 30

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 31

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 31

caggctgtgc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacgtcgga aataatgctg taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact cctcatctat tatgatgatc tgctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgaag tgcagctggt gcagtctgga 420

gcagaggtga aaaagcccgg ggagtctctg aagatctcct gtaagggttc tggatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtc 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttcta cttgggactc tatgtacatg 720

gattactggg gtcaaggtac tctggtgacc gtctcctca 759

<210> 32

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 32

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 33

<211> 756

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 33

cagcctgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatc 60

acctgtggag gaaacaacat tggaagtgaa agtgtgcact ggtaccagca gaagccaggc 120

caggccccta tggtggtcat ctattatgat agcaaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgaccg tcagcagggt cgaagccgag 240

gatgaggccg actattactg tcaggtgtgg aatagtagta gtgatcatcg aggagtgttc 300

ggcgggaggga ccaagctgac cgtcctaggt tctagaggtg gtggtggtag cggcggcggc 360

ggctctggtg gtggtggatc cctcgagatg gccgaagtgc agctggtgca gtctggagca 420

gaggtgaaaa agcccgggga gtctctgagg atctcctgta agggttctgg atacagcttt 480

accagctact ggatcggctg ggtgcgccag atgcccggga aaggcctgga gtggatgggg 540

atcatctatc ctggtgactc tgataccaga tacagcccgt ccttccaagg ccaggtcacc 600

atctcagccg acaagtccat cagcaccgcc tacctgcagt ggagcagcct gaaggcctcg 660

gacaccgcca tgtattactg tgcgcgcgtt acttactcta tggactctta ctacttcgat 720

tcttggggtc aaggtactct ggtgaccgtc tcctca 756

<210> 34

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 34

Gln Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Glu Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Met Val Val Ile Tyr

35 40 45

Tyr Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Val Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asn Ser Ser Ser Asp His

85 90 95

Arg Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu

115 120 125

Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

130 135 140

Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

145 150 155 160

Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu

165 170 175

Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser

180 185 190

Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

195 200 205

Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met

210 215 220

Tyr Tyr Cys Ala Arg Val Thr Tyr Ser Met Asp Ser Tyr Tyr Phe Asp

225 230 235 240

Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 35

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 35

ctgcctgtgc ttactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcaacagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ctggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac tgtctcctca 780

<210> 36

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 36

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Asn Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 37

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 37

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtatttggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcggc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgcaagg gttctggata cagctttacc 480

agcttctgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgcgatgt attactgtgt gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtaac cgtctcctca 780

<210> 38

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 38

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Phe Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Val Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 39

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 39

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggca gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 40

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 40

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Glu

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 41

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 41

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggcggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaactt ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 42

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 42

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Leu Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 43

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 43

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accatgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gtgctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 44

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 44

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Met Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ala Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 45

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 45

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacatggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 46

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 46

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Met Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 47

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 47

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcaaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgagtatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgtcaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 48

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 48

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Glu Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 49

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 49

ctgcctgtgc tgactcagcc accctcgatg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtggttatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 50

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 50

Leu Pro Val Leu Thr Gln Pro Ser Met Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Gly Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 51

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 51

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctacgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtacta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtt tctgaagatc tcctgtaagg gttctggata cagcttcacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcgccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 52

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 52

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Thr Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Phe Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Ala Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 53

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 53

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaccactgcc accctgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tagtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactccga taccagatac agcccgtcct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gaacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 54

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 54

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Thr Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Asn Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 55

<211> 780

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 55

ctgcctgtgc tgactcagcc accctcggtg tcagtggccc caggaaagac ggccaggatt 60

acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120

caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accttgacca tcagcagggt cgaagccggg 240

gatgaggccg actattactg tcaggtgtgg gatagtagta gtgattatgt ggtattcggc 300

ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagtgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc gaagtgcagc tggtgcagtc tggagcagag 420

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagctttacc 480

agctactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 540

atctatcctg gtgactctga taccagatac agcccgttct tccaaggcca ggtcaccatc 600

tcagccgaca agtccatcag caccgcctac ctgcagtgga gcagcctgaa ggcctcggac 660

accgccatgt attactgtgc gcgccaggtt tggggttggc agggtggtat gtacccgcgt 720

tctaactggt ggtacaacat ggattcttgg ggtcaaggta ctctggtgac cgtctcctca 780

<210> 56

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 56

Leu Pro Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp Tyr

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

130 135 140

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr

145 150 155 160

Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

165 170 175

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro

180 185 190

Phe Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

195 200 205

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

210 215 220

Tyr Cys Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg

225 230 235 240

Ser Asn Trp Trp Tyr Asn Met Asp Ser Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ser

260

<210> 57

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 57

caggctgtgc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacgtcgga aataatgctg taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact cctcatctat tatgatgatc tgctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgaag tgcagctggt gcagtccgga 420

gcagaggtga aaaagcccgg ggagtctctg aagatctcct gtaagggttc tggatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtc 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttctt cttgggactc tatgtacatg 720

gattactggg gtcaaggtac tatggtgacc gtctcctca 759

<210> 58

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 58

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Ser Trp Asp Ser Met Tyr Met

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

245 250

<210> 59

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 59

caggctgtgc ttactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaaacagctc caacgtcgga aataatgcta taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact cctcatctat tatgatgatc tgctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgaag tgcagctggt gcagtctgga 420

gcagaggtgg aaaagcccgg ggagtctctg aagatctcct gtaagggttc tggatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtc 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttcta cttgggactc tatgtacatg 720

gaatactggg gtcaaggtac tctggtgacc gtctcctca 759

<210> 60

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 60

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Asn Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Ile Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu

130 135 140

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met

225 230 235 240

Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 61

<211> 760

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 61

caggctgtgc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacgtcgga aataatgctg taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact cctcatctat tatgatgatc tgctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgaag tgcagctggt gcagtctgga 420

gcagaggtga aaaagcccgg ggggtctctg aagatctcct gtaagggttc tggatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtc 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttcta cttgggactc tatgtacatg 720

gattactggg gtcaaggtac tctggtgacc gtctcctcaa 760

<210> 62

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 62

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Gly Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 63

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 63

caggctgtgc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacgtcgga aataatgctg taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact cctcatctat tatgatgatc agctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgaag tgcagctggt gcagtctgga 420

gcagaggtga aaaagcccgg ggagtctctg aagatctcct gtaagggttc tggatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtc 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttcta cttgggactc tttgtacatg 720

gattactggg gtcaaggtac tctggtgacc gtctcctca 759

<210> 64

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 64

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Gln Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Thr Trp Asp Ser Leu Tyr Met

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 65

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 65

caggctgtgc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaaacagctc caacgtcgga aataatgctg taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact tctcatctat tatgatgatc tgctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgaag tgcagctggt gcagtctgga 420

gcagaggtga aaaagcccgg ggagtctctg aagatctcct gtaagggttc tggatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtc 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttcta cttgggactc tatgtacatg 720

gattactggg gtcaaggtac tctggtgacc gtctcctca 759

<210> 66

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 66

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Asn Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 67

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 67

caggctgtgc tgactcagcc accctcggtg tccgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacgtcgga aataatgctg taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact cctcatctat tatgatgatc tgctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg ttccctcgag atggccgaag tacagctggt gcagtcagga 420

gcagaggtga aaaagcccgg ggaatctctg aagatctcct gtaagggttc tgcatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtc 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttcta cttgggactc tatgtacatg 720

gattactggg gtctaggtac tctggtgacc gtctcctca 759

<210> 68

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 68

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Ala Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met

225 230 235 240

Asp Tyr Trp Gly Leu Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 69

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 69

caggctgtgc tgactcaacc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacgtcgga aataatgctg taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact cctcatctat tatgatgatc agctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcggg atggccgaag tgcagctggt gcagtctgga 420

gcagaggtga aaaagcccgg ggagtctctg aagatctcct gtaagggttc tggatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtt 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttcta cttgggactc tatgtacatg 720

gattactggg gtcaaggtac tctggtaacc gtctcctca 759

<210> 70

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 70

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Gln Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Gly Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 71

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 71

caggctgtgc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacgtcggt aataatgctg taaactggta ccagcaggtc 120

cgggaaagg ctcccaaact cctcatctat tatgatgatc tgctgtcctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggccgatta ttattgtgca gcatgggatg acagcctgaa tggtccggta 300

ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggccgaag tgcagctggt gcagtctgga 420

gcagaggtga aaaagcccgg ggagtctctg aagatctcct gtaagggttc tggatacagc 480

tttaccagct actggatcgg ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540

gggatcatct atcctggtga ctctgatacc agatacagcc cgtccttcca aggccaggtc 600

accatctcag ccgacaagtc catcagcacc gcctacctgc agtggagcag cctgaaggcc 660

tcggacaccg ccatgtatta ctgtgcgcgc tggtcttctt cttgggactc tatgtacatg 720

gattactggg gtcaaggtac tctggtgacc gtctcctca 759

<210> 72

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 72

Gln Ala Val Leu Thr Gln Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Val Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Ser Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

145 150 155 160

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

165 170 175

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

180 185 190

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

195 200 205

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Trp Ser Ser Ser Trp Asp Ser Met Tyr Met

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 73

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 73

Gly Phe Thr Phe Ser Ser Tyr Ala

fifteen

<210> 74

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 74

Ile Ser Gly Ser Gly Gly Ser Thr

fifteen

<210> 75

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 75

Ala Arg Tyr Tyr Tyr Ser Arg Leu Asp Tyr

1 5 10

<210> 76

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 76

Gly Phe Thr Phe Ser Ser Tyr Ala

fifteen

<210> 77

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 77

Ile Ser Ala Ser Gly Gly Ser Thr

fifteen

<210> 78

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 78

Ala Arg Tyr Tyr Leu Ser Gln Ile Asp Ser

1 5 10

<210> 79

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 79

Gly Tyr Thr Phe Thr Asp Tyr Tyr

fifteen

<210> 80

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 80

Val Asp Pro Glu Asp Gly Glu Thr

fifteen

<210> 81

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 81

Ala Thr Gly Ile Tyr Ser Arg Pro Leu Gly Tyr

1 5 10

<210> 82

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 82

Gly Phe Thr Phe Ser Ser Tyr Ala

fifteen

<210> 83

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 83

Ile Ser Gly Ser Gly Gly Ser Thr

fifteen

<210> 84

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 84

Ala Arg Ser Asp Gly Lys His Phe Trp Gln Gln Tyr Asp Ala

1 5 10

<210> 85

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 85

Gly Phe Thr Val Ser Ser Asn Tyr

fifteen

<210> 86

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 86

Ile Ser Gly Ser Gly Gly Ser Thr

fifteen

<210> 87

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 87

Ala Arg Met Asn Ile Asp Tyr

fifteen

<210> 88

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 88

Glu Tyr Ser Phe Ala Ser Tyr Trp

fifteen

<210> 89

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 89

Ile Asp Pro Ser Asp Ser Tyr Thr

fifteen

<210> 90

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 90

Ala Arg Pro Phe Gln Tyr Asp Tyr Gly Gly Tyr Ser Asp Ala Phe Asp

1 5 10 15

ile

<210> 91

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 91

Gly Gly Thr Phe Ser Ser Ser Ser

fifteen

<210> 92

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 92

Ile Ile Pro Ile Val Gly Thr Pro

fifteen

<210> 93

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 93

Ala Arg Gly Gly Tyr Arg Asp Tyr Met Asp Val

1 5 10

<210> 94

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 94

Gly Phe Thr Val Ser Ser Asn Tyr

fifteen

<210> 95

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 95

Ile Tyr Ser Gly Gly Ser Thr

fifteen

<210> 96

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 96

Ala Arg Gly Gly Phe Gly Ala Glu Phe Asp Tyr

1 5 10

<210> 97

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 97

Gly Phe Thr Val Ser Ser Asn Tyr

fifteen

<210> 98

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 98

Ile Tyr Ser Gly Gly Ser Thr

fifteen

<210> 99

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 99

Ala Arg Gly Gly Ile Ser Asp Asp Tyr Tyr Gly Ser Gly Ser Tyr Asp

1 5 10 15

Asn

<210> 100

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 100

Gly Phe Thr Val Ser Ser Asn Tyr

fifteen

<210> 101

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 101

Ile Tyr Ser Gly Gly Ser Thr

fifteen

<210> 102

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 102

Ala Arg Glu Arg Gly Met Gly Tyr Ala Phe Asp Ile

1 5 10

<210> 103

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 103

Gly Val Ser Met Ser Glu Asn Tyr

fifteen

<210> 104

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 104

Ala His Tyr Thr Gly Asp Thr

fifteen

<210> 105

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 105

Ala Ser Tyr His Pro Phe Asn Tyr

fifteen

<210> 106

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 106

Gly Tyr Thr Phe Asn Asp Phe Tyr

fifteen

<210> 107

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 107

Ile Asp Pro Glu Asp Gly Lys Thr

fifteen

<210> 108

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 108

Thr Thr Asp Trp Gly Tyr Ser Ser Ser Leu Arg Glu Glu Asp Ile Trp

1 5 10 15

Tyr Asp Cys

<210> 109

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 109

Gly Gly Thr Phe Ser Ser Tyr Ala

fifteen

<210> 110

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 110

Ile Ile Pro Ile Phe Gly Thr Ala

fifteen

<210> 111

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 111

Ala Arg Asp Tyr Gly Tyr Gly Asp Tyr Gly Asp Ala Phe Asp Ile

1 5 10 15

<210> 112

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 112

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 113

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 113

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 114

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 114

Ala Arg Val Val Gly Thr Ile Tyr Ser Met Gln Tyr Asp Val

1 5 10

<210> 115

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 115

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 116

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 116

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 117

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 117

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 118

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 118

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 119

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 119

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 120

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 120

Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met Asp Tyr

1 5 10

<210> 121

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 121

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 122

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 122

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 123

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 123

Ala Arg Val Thr Tyr Ser Met Asp Ser Tyr Tyr Phe Asp Ser

1 5 10

<210> 124

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 124

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 125

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 125

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 126

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 126

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 127

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 127

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 128

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 128

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 129

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 129

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 130

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 130

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 131

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 131

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 132

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 132

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 133

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 133

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 134

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 134

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 135

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 135

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 136

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 136

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 137

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 137

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 138

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 138

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 139

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 139

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 140

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 140

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 141

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 141

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Asn Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 142

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 142

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 143

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 143

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 144

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 144

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 145

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 145

Gly Tyr Ser Phe Thr Ser Phe Trp

fifteen

<210> 146

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 146

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 147

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 147

Val Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 148

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 148

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 149

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 149

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 150

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 150

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 151

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 151

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 152

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 152

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 153

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 153

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Leu Asp Ser

twenty

<210> 154

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 154

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 155

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 155

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 156

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 156

Ala Arg Gln Val Trp Gly Trp Gln Gly Gly Met Tyr Pro Arg Ser Asn

1 5 10 15

Trp Trp Tyr Asn Met Asp Ser

twenty

<210> 157

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 157

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 158

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 158

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 159

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 159

Ala Arg Trp Ser Ser Ser Trp Asp Ser Met Tyr Met Asp Tyr

1 5 10

<210> 160

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 160

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 161

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 161

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 162

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 162

Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met Glu Tyr

1 5 10

<210> 163

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 163

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 164

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 164

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 165

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 165

Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met Asp Tyr

1 5 10

<210> 166

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 166

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 167

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 167

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 168

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 168

Ala Arg Trp Ser Ser Thr Trp Asp Ser Leu Tyr Met Asp Tyr

1 5 10

<210> 169

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 169

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 170

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 170

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 171

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 171

Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met Asp Tyr

1 5 10

<210> 172

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 172

Ala Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 173

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 173

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 174

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 174

Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met Asp Tyr

1 5 10

<210> 175

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 175

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 176

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 176

Ile Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 177

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 177

Ala Arg Trp Ser Ser Thr Trp Asp Ser Met Tyr Met Asp Tyr

1 5 10

<210> 178

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 178

Gly Tyr Ser Phe Thr Ser Tyr Trp

fifteen

<210> 179

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 179

Tyr Pro Gly Asp Ser Asp Thr

fifteen

<210> 180

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 180

Ala Arg Trp Ser Ser Ser Trp Asp Ser Met Tyr Met Asp Tyr

1 5 10

<210> 181

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 181

Ser Ser Asn Ile Gly Asn Asn Tyr

fifteen

<210> 182

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 182

Asp Asn Asn

one

<210> 183

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 183

Gly Thr Trp Asp Ser Ser Leu Ser Ala Gly Val

1 5 10

<210> 184

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 184

Ser Ser Asn Ile Gly Asn Asn Tyr

fifteen

<210> 185

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 185

Glu Asn Asn

one

<210> 186

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 186

Gly Thr Trp Asp Ser Ser Leu Arg Ala Gly Val

1 5 10

<210> 187

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 187

Ser Ser Asn Ile Gly Ser Asn Thr

fifteen

<210> 188

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 188

Ser Asn Asn

one

<210> 189

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 189

Ala Ala Trp Asp Asp Ser Leu Asn Gly His Val Val

1 5 10

<210> 190

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 190

Ser Ser Asn Ile Gly Ser His Thr

fifteen

<210> 191

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 191

Ser Asn Asn

one

<210> 192

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 192

Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val

1 5 10

<210> 193

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 193

Gln Gly Ile Thr Asn Ser

fifteen

<210> 194

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 194

Ala Ala Ser

one

<210> 195

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 195

Gln His Tyr Leu Gly Thr Pro Tyr Ser

fifteen

<210> 196

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 196

Gln Ser Val Ser Arg Phe

fifteen

<210> 197

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 197

Gly Val Ser

one

<210> 198

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 198

Gln Glu Ser Tyr Ile Ile Pro Leu Thr

fifteen

<210> 199

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 199

Gln Ser Leu Leu Asp Ser Asn Gly Phe Asn Ser

1 5 10

<210> 200

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 200

Leu Gly Ser

one

<210> 201

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 201

Met Gln Ser Leu Gln Ile Pro Thr

fifteen

<210> 202

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 202

Ser Ser Asn Ile Gly Ser Asn Tyr

fifteen

<210> 203

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 203

Arg Asn Asn

one

<210> 204

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 204

Ala Ala Trp Asp Asp Ser Leu Ser Gly Tyr Val

1 5 10

<210> 205

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 205

Lys Leu Gly Asp Lys Tyr

fifteen

<210> 206

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 206

Gln Asp Asn

one

<210> 207

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 207

Gln Ala Trp Asp Ser Ser Thr Glu Asp Val

1 5 10

<210> 208

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 208

Gln Ser Ile Ser Ser Tyr

fifteen

<210> 209

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 209

Ala Ala Ser

one

<210> 210

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 210

Gln Gln Ser Tyr Ser Thr Pro Phe Thr

fifteen

<210> 211

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 211

Glyn Gly Ile Gly Ser Tyr

fifteen

<210> 212

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 212

Pro Ala Ser

one

<210> 213

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 213

Gln Gln Leu Asn Ser Leu

fifteen

<210> 214

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 214

Ser Ser Asn Ile Gly Thr Lys Thr

fifteen

<210> 215

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 215

Ser Asn Tyr

one

<210> 216

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 216

Ala Leu Trp Asp Asp Ser Leu Asp Gly Tyr Val

1 5 10

<210> 217

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 217

Asn Ile Gly Ser Lys Ser

fifteen

<210> 218

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 218

Tyr Asp Ser

one

<210> 219

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 219

Gln Val Trp Asp Ser Ser Ser Asp His Tyr Val

1 5 10

<210> 220

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 220

Asn Ile Gly Ser Lys Ser

fifteen

<210> 221

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 221

Asp Asp Ser

one

<210> 222

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 222

Gln Val Trp Asp Ser Ser Ser Asp His Tyr Val

1 5 10

<210> 223

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 223

Asn Ile Gly Ser Lys Ser

fifteen

<210> 224

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 224

Asp Asp Ser

one

<210> 225

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 225

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 226

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 226

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 227

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 227

Tyr Asp Asp

one

<210> 228

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 228

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 229

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 229

Asn Ile Gly Ser Glu Ser

fifteen

<210> 230

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 230

Tyr Asp Ser

one

<210> 231

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 231

Gln Val Trp Asn Ser Ser Ser Asp His Arg Gly Val

1 5 10

<210> 232

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 232

Asn Ile Gly Ser Lys Ser

fifteen

<210> 233

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 233

Asp Asp Ser

one

<210> 234

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 234

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 235

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 235

Asn Ile Gly Ser Lys Ser

fifteen

<210> 236

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 236

Asp Asp Ser

one

<210> 237

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 237

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 238

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 238

Asn Ile Gly Ser Lys Ser

fifteen

<210> 239

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 239

Asp Asp Ser

one

<210> 240

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 240

Gln Val Trp Asp Ser Ser Ser Glu Tyr Val Val

1 5 10

<210> 241

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 241

Asn Ile Gly Ser Lys Ser

fifteen

<210> 242

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 242

Asp Asp Ser

one

<210> 243

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 243

Gln Val Trp Asp Ser Ser Ser Gly Tyr Val Val

1 5 10

<210> 244

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 244

Asn Ile Gly Ser Lys Ser

fifteen

<210> 245

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 245

Asp Asp Ser

one

<210> 246

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 246

Gln Val Trp Asp Ser Thr Ser Asp Tyr Val Val

1 5 10

<210> 247

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 247

Asn Ile Gly Ser Lys Ser

fifteen

<210> 248

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 248

Asp Asp Ser

one

<210> 249

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 249

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 250

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 250

Asn Ile Gly Ser Lys Ser

fifteen

<210> 251

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 251

Asp Asp Ser

one

<210> 252

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 252

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 253

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 253

Asn Ile Gly Ser Lys Ser

fifteen

<210> 254

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 254

Asp Asp Ser

one

<210> 255

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 255

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 256

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 256

Asn Ile Gly Ser Lys Ser

fifteen

<210> 257

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 257

Asp Asp Ser

one

<210> 258

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 258

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 259

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 259

Asn Ile Gly Ser Lys Ser

fifteen

<210> 260

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 260

Asp Asp Ser

one

<210> 261

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 261

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 262

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 262

Asn Ile Gly Ser Lys Ser

fifteen

<210> 263

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 263

Asp Asp Ser

one

<210> 264

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 264

Gln Val Trp Asp Ser Ser Ser Asp Tyr Val Val

1 5 10

<210> 265

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 265

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 266

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 266

Tyr Asp Asp

one

<210> 267

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 267

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 268

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 268

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 269

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 269

Tyr Asp Asp

one

<210> 270

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 270

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 271

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 271

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 272

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 272

Tyr Asp Asp

one

<210> 273

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 273

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 274

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 274

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 275

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 275

Tyr Asp Asp

one

<210> 276

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 276

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 277

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 277

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 278

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 278

Tyr Asp Asp

one

<210> 279

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 279

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 280

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 280

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 281

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 281

Tyr Asp Asp

one

<210> 282

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 282

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 283

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 283

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 284

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 284

Tyr Asp Asp

one

<210> 285

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 285

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 286

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 286

Ser Ser Asn Val Gly Asn Asn Ala

fifteen

<210> 287

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 287

Tyr Asp Asp

one

<210> 288

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (4)..(7)

<223> Xaa can be any naturally occurring amino acids

<400> 288

Ile Xaa Pro Xaa Xaa Xaa Xaa Thr

fifteen

<210> 289

<211> 556

<212> PRT

<213> Homo sapeins

<400> 289

Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met

1 5 10 15

Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp

20 25 30

Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln

35 40 45

Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu

50 55 60

Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile

65 70 75 80

Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu

85 90 95

Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr

100 105 110

Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp

115 120 125

Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro

130 135 140

Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala

145 150 155 160

Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro

165 170 175

Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro

180 185 190

Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser

195 200 205

Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser

210 215 220

Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp

225 230 235 240

Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala

245 250 255

Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu

260 265 270

Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly

275 280 285

Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu

290 295 300

Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg

305 310 315 320

Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val

325 330 335

Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu

340 345 350

Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala

355 360 365

Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp

370 375 380

Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly

385 390 395 400

Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Pro Asp Ser Glu Glu

405 410 415

Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu

420 425 430

Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly

435 440 445

Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu

450 455 460

Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser

465 470 475 480

Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly

485 490 495

Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln

500 505 510

Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala

515 520 525

Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp

530 535 540

Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg

545 550 555

<210> 290

<211> 488

<212> PRT

<213> Homo sapiens

<400> 290

Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met

1 5 10 15

Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp

20 25 30

Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln

35 40 45

Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu

50 55 60

Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile

65 70 75 80

Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu

85 90 95

Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr

100 105 110

Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp

115 120 125

Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro

130 135 140

Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala

145 150 155 160

Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro

165 170 175

Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro

180 185 190

Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser

195 200 205

Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser

210 215 220

Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp

225 230 235 240

Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala

245 250 255

Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu

260 265 270

Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly

275 280 285

Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu

290 295 300

Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg

305 310 315 320

Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val

325 330 335

Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu

340 345 350

Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala

355 360 365

Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp

370 375 380

Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly

385 390 395 400

Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Pro Asp Ser Glu Glu

405 410 415

Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu

420 425 430

Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly

435 440 445

Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu

450 455 460

Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser

465 470 475 480

Pro His Gly Ser Ala Trp Asp Pro

485

<210> 291

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<220>

<221> misc_feature

<222> (2)..(8)

<223> X2 is F, G, Y or V, X3 is S or T, X5 is S or T, X7 is N or Y, and

X8 is A, W or Y

<400> 291

Gly Xaa Xaa Phe Xaa Ser Xaa Xaa

fifteen

<210> 292

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<220>

<221> misc_feature

<222> (2)..(7)

<223> X2 is S, D or Y, X4 is E, S, G or I, X5 is D, F or V, X6 is G or

S, and X7 is K, E, Y, D or T

<400> 292

Ile Xaa Pro Xaa Xaa Xaa Xaa Thr

fifteen

<210> 293

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<220>

<221> misc_feaure

<222> (4)..(8)

<223> X4 is I or V, X6 is N, S or T, X7 is N, H or K, X8 is Y, A or T

<400> 293

Ser Ser Asn Xaa Gly Xaa Xaa Xaa

fifteen

<210> 294

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<220>

<221> misc_feature

<222> (5)..(5)

<223> X5 is K or E

<400> 294

Asn Ile Gly Ser Xaa Ser

fifteen

<210> 295

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<220>

<221> misc_feature

<222> (1)..(3)

<223> X1 is D, E, S, R or Y, X2 is N or D, X3 is N, Y, S or D

<400> 295

Xaa Xaa Xaa

one

<210> 296

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 296

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 297

<211> 6

<212> PRT

<213> Mus musculus

<400> 297

Gln Asp Ile Ser Lys Tyr

fifteen

<210> 298

<211> 3

<212> PRT

<213> Mus musculus

<400> 298

His Thr Ser

one

<210> 299

<211> 9

<212> PRT

<213> Mus musculus

<400> 299

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

fifteen

<210> 300

<211> 8

<212> PRT

<213> Mus musculus

<400> 300

Gly Val Ser Leu Pro Asp Tyr Gly

fifteen

<210> 301

<211> 7

<212> PRT

<213> Mus musculus

<400> 301

Ile Trp Gly Ser Glu Thr Thr

fifteen

<210> 302

<211> 14

<212> PRT

<213> Mus musculus

<400> 302

Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<---

Claims (124)

1. An antibody or antigen-binding fragment thereof that specifically binds human CD19, wherein said antibody or antigen-binding fragment includes heavy chain CDR1 (HC-CDR1), heavy chain CDR2 (HC-CDR2) and heavy chain CDR3 (HC-CDR3) and light chain CDR1 chain (LC-CDR1), light chain CDR2 (LC-CDR2) and light chain CDR3 (LC-CDR3), where HC-CDR1 includes G-X1-X2-F-X3-S-X4-X5 (SEQ ID NO: 291) and where X1 is F, G, Y or V, X2 is S or T, X3 is S or T, X4 is N or Y and X5 is A, W or Y; where HC-CDR2 includes I-X6-P-X7-X8-X9-X10-T (SEQ ID NO: 292) and where X6 is S, D or Y, X7 is E, S, G or I, X8 is D, F or V, X9 is G or S and X10 is K, E, Y, D or T; where HC-CDR3 is selected from SEQ ID NO: 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 211, 114, 117, 120 or 123, where LC-CDR1 includes S-S-N-X11-G-X12-X13-X14 (SEQ ID NO: 293) or N-I-G-S-X15-S (SEQ ID NO: 294) and where X11 is I or V, X12 is N, S or T, X13 is N, H or K, X14 is Y, A or T and X15 is K or E; and/or where LC-CDR2 includes X16-X17-X18 (SEQ ID NO: 295) and where X16 is D, E, S, R or Y, X 17 is N or D and X18 is N, Y, S or D, where LC-CDR3 is selected from SEQ ID NO: 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228 or 231. 2. An antibody or antigen-binding fragment thereof according to claim 1, wherein said antibody or antigen-binding fragment comprises: at least one heavy chain CDR (HC-CDR) containing a sequence that is at least 95% identical to any of the sequences of SEQ ID NOs: 73-123, and/or at least one CDR (LC-CDR) light chain containing a sequence that is at least 95% identical to any of the sequences of SEQ ID NO: 181-231. 3. An antibody or antigen-binding fragment thereof according to claim 2, wherein said antibody or antigen-binding fragment comprises: (a) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each of which has a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO : 183 respectively; (b) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 184, SEQ ID NO: 185, and SEQ ID NO : 186 respectively; (c) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO : 189 respectively; (d) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO. : 192 respectively; (e) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO : 195 respectively; (f) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO : 198 respectively; (g) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO 201 respectively; (h) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each of which has a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 202, SEQ ID NO: 203, and SEQ ID NO : 204 respectively; (i) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO : 207 respectively; (j) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 208, SEQ ID NO: 209, and SEQ ID NO : 210 respectively; (k) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO : 213 respectively; (1) HC-CDR1, HC-CDR2, and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences set forth in SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO : 216 respectively; (m) HC-CDR1, HC-CDR2, and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each of which has a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO : 219 respectively; (n) HC-CDR1, HC-CDR2, and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO : 222 respectively; (o) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 223, SEQ ID NO: 224, and SEQ ID NO : 225 respectively; (p) HC-CDR1, HC-CDR2 and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each of which has a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 226, SEQ ID NO: 227, and SEQ ID NO : 228 respectively; or (q) HC-CDR1, HC-CDR2, and HC-CDR3 each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively, and LC-CDR1, LC-CDR2, and LC-CDR3, each having a sequence that is at least 95% identical to the amino acid sequences shown in SEQ ID NO: 229, SEQ ID NO: 230, and SEQ ID NO : 231 respectively. 4. An antibody or antigen-binding fragment thereof according to claim 3, wherein said antibody or antigen-binding fragment comprises: (a) HC-CDRs from SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75 and LC-CDRs from SEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183; (b) HC-CDRs from SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78 and LC-CDRs from SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186; (c) HC-CDRs from SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81 and LC-CDRs from SEQ ID NO: 187, SEQ ID NO: 188 and SEQ ID NO: 189; (d) HC-CDRs from SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84 and LC-CDRs from SEQ ID NO: 190, SEQ ID NO: 191 and SEQ ID NO: 192; (e) HC-CDRs from SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87 and LC-CDRs from SEQ ID NO: 193, SEQ ID NO: 194 and SEQ ID NO: 195; (f) HC-CDRs from SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90 and LC-CDRs from SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198; (g) HC-CDRs from SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93 and LC-CDRs from SEQ ID NO: 199, SEQ ID NO: 200 and SEQ ID NO 201; (h) HC-CDRs from SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96 and LC-CDRs from SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204; (i) HC-CDRs from SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99 and LC-CDRs from SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207; (j) HC-CDRs from SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102 and LC-CDRs from SEQ ID NO: 208, SEQ ID NO: 209 and SEQ ID NO: 210; (k) HC-CDRs from SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105 and LC-CDRs from SEQ ID NO: 211, SEQ ID NO: 212 and SEQ ID NO: 213; (l) HC-CDRs from SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108 and LC-CDRs from SEQ ID NO: 214, SEQ ID NO: 215 and SEQ ID NO: 216; (m) HC-CDRs from SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111 and LC-CDRs from SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219; (n) HC-CDRs from SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114 and LC-CDRs from SEQ ID NO: 220, SEQ ID NO: 221 and SEQ ID NO: 222; (o) HC-CDRs from SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, and LC-CDRs from SEQ ID NO: 223, SEQ ID NO: 224, and SEQ ID NO: 225; (p) HC-CDRs from SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120 and LC-CDRs from SEQ ID NO: 226, SEQ ID NO: 227 and SEQ ID NO: 228; or (q) HC-CDRs from SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123 and LC-CDRs from SEQ ID NO: 229, SEQ ID NO: 230 and SEQ ID NO: 231. 5 An antibody or antigen-binding fragment according to any one of paragraphs. 2-4, wherein said antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) having a sequence that is at least 95% identical to HCVR of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34 and a light chain variable region (LCVR) having a sequence that is at least 95% identical to the LCVR of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34. 6. The antibody or antigen-binding fragment of claim 5, wherein said antibody or antigen-binding fragment includes: (a) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 2 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 2; (b) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 4 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 4; (c) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 6 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 6; (d) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 8 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 8; (e) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 10 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 10; (f) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 12 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 12; (g) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 14 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 14; (h) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 16 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 16; (i) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 18 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 18; (j) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 20 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 20; (k) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 22 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 22; (1) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 24 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 24; (m) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 26 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 26; (n) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 28 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 28; (o) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 30 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 30; (p) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 32 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 32; or (q) HCVR having a sequence that is at least 95% identical to HCVR of SEQ ID NO: 34 and LCVR having a sequence that is at least 95% identical to LCVR of SEQ ID NO: 34. 7. An antibody or antigen-binding fragment thereof according to claim 5 or 6, wherein said antibody or antigen-binding fragment comprises the sequences HCVR and LCVR of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 34. 8. An antibody or antigen-binding fragment according to any one of paragraphs. 1-7, wherein said antibody or antigen-binding fragment further comprises one to five amino acid substitutions in the HC-CDR, HC-framework, LC-CDR, LC-framework, or combinations thereof. 9. An antibody or antigen-binding fragment thereof according to claim 8, wherein said antibody or antigen-binding fragment includes: light chain variable region with amino acid substitutions D52N and D95E, heavy chain variable region with amino acid substitution S63F, heavy chain variable region with amino acid substitutions Q3R, Y32F and A97V, heavy chain variable region with amino acid substitution M117L or a light chain variable region with amino acid substitutions S25N and V34I; and a heavy chain variable region with amino acid substitutions K12E and D109E. 10. An antibody or antigen-binding fragment thereof according to claim 8, wherein said antibody or antigen-binding fragment includes: (a) HC-CDRs from SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126 and LC-CDRs from SEQ ID NO: 232, SEQ ID NO: 233 and SEQ ID NO: 234; (b) HC-CDRs from SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129 and LC-CDRs from SEQ ID NO: 235, SEQ ID NO: 236 and SEQ ID NO: 237; (c) HC-CDRs from SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132 and LC-CDRs from SEQ ID NO: 238, SEQ ID NO: 239 and SEQ ID NO: 240; (d) HC-CDRs from SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135 and LC-CDRs from SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 243; (e) HC-CDRs from SEQ ID NO: 136, SEQ ID NO: 137 and SEQ ID NO: 138 and LC-CDRs from SEQ ID NO: 244, SEQ ID NO: 245 and SEQ ID NO: 246; (f) HC-CDRs from SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141 and LC-CDRs from SEQ ID NO: 247, SEQ ID NO: 248 and SEQ ID NO: 249; (g) HC-CDRs from SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144 and LC-CDRs from SEQ ID NO: 250, SEQ ID NO: 251 and SEQ ID NO: 252; (h) HC-CDRs from SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 147 and LC-CDRs from SEQ ID NO: 253, SEQ ID NO: 254 and SEQ ID NO: 255; (i) HC-CDRs from SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150 and LC-CDRs from SEQ ID NO: 256, SEQ ID NO: 257 and SEQ ID NO: 258; (j) HC-CDRs from SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153 and LC-CDRs from SEQ ID NO: 259, SEQ ID NO: 260 and SEQ ID NO: 261; (k) HC-CDRs from SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156 and LC-CDRs from SEQ ID NO: 262, SEQ ID NO: 263 and SEQ ID NO: 264; (l) HC-CDRs from SEQ ID NO: 157, SEQ ID NO: 158 and SEQ ID NO: 159 and LC-CDRs from SEQ ID NO: 265, SEQ ID NO: 266 and SEQ ID NO: 267; (m) HC-CDRs from SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 163 and LC-CDRs from SEQ ID NO: 268, SEQ ID NO: 269 and SEQ ID NO: 270; (n) HC-CDRs from SEQ ID NO: 163, SEQ ID NO: 164 and SEQ ID NO: 165 and LC-CDRs from SEQ ID NO: 271, SEQ ID NO: 272 and SEQ ID NO: 273; (o) HC-CDRs from SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168 and LC-CDRs from SEQ ID NO: 274, SEQ ID NO: 275 and SEQ ID NO: 276; (p) HC-CDRs from SEQ ID NO: 169, SEQ ID NO: 170 and SEQ ID NO: 171 and LC-CDRs from SEQ ID NO: 277, SEQ ID NO: 278 and SEQ ID NO: 279; (q) HC-CDRs from SEQ ID NO: 172, SEQ ID NO: 173 and SEQ ID NO: 174 and LC-CDRs from SEQ ID NO: 280, SEQ ID NO: 281 and SEQ ID NO: 282; (r) HC-CDRs from SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177 and LC-CDRs from SEQ ID NO: 283, SEQ ID NO: 284 and SEQ ID NO: 285; or (s) HC-CDRs from SEQ ID NO: 178, SEQ ID NO: 179 and SEQ ID NO: 180 and LC-CDRs from SEQ ID NO: 286, SEQ ID NO: 287 and SEQ ID NO: 288. 11. The antibody or antigen-binding fragment of claim 8, wherein said antibody or antigen-binding fragment comprises the sequences HCVR and LCVR of any of SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72. 12. An antibody or antigen-binding fragment thereof according to claim 8, whose sequence includes 1-5 amino acid substitutions compared to the parent antibody having HCVR and LCVR of SEQ ID NO: 30 or SEQ ID NO: 32. 13. An antibody or antigen-binding fragment thereof according to claim 12, where 1-5 amino acid substitutions are in the framework region and/or CDR. 14. An antibody or antigen-binding fragment according to any one of paragraphs. 1-13 which is or includes an immunoglobulin, Fab, F(ab')2, Fd, Fv, single chain Fv (scFv) or dAb. 15. An antibody or antigen-binding fragment according to any one of paragraphs. 1-14, wherein said antibody or antigen binding fragment is conjugated to a therapeutic agent, detection agent, cytotoxic agent, or fragment or radioisotope. 16. An antibody or antigen-binding fragment according to any one of paragraphs. 1-15, wherein the antibody or antigen-binding fragment is based on a human antibody. 17. A bispecific antibody that specifically binds CD19 and a second antigen, comprising a first antigen-binding site and a second antigen-binding site, characterized in that the first antigen-binding site originates from or is based on an antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-16. 18. A bispecific antibody according to claim 17, characterized in that the second antigen-binding site binds an immune cell selected from the group consisting of a T cell, NK cell, B cell, dendritic cell, monocyte, macrophage, neutrophil, mesenchymal stem cell and neuronal stem cells. 19. A chimeric antigen receptor that specifically binds human CD19 and contains an antigen-binding site of an antibody or antigen-binding fragment according to any one of paragraphs. 1-16. 20. The chimeric antigen receptor of claim 19, further comprising a transmembrane domain and an intracellular signaling domain, the intracellular signaling domain of which includes a CD3ζ intracellular signal sequence and a CD28 intracellular signal sequence. 21. A chimeric antigen receptor according to claim 19, further comprising a transmembrane domain and/or an intracellular signaling domain of a native cell receptor. 22. The chimeric antigen receptor of claim 21, wherein the native cell receptor is the T cell receptor (TCR). 23. A nucleic acid molecule encoding an antibody or antigen-binding fragment according to any one of paragraphs. 1-16, a bispecific antibody according to claim 17 or 18, or chimeric antigen receptor according to any one of paragraphs. 19-22. 24. A cell comprising a nucleic acid molecule according to claim 23 and expressing an antibody or antigen-binding fragment according to any one of paragraphs. 1-16, a bispecific antibody according to claim 17 or 18, or a chimeric antigen receptor according to any one of claims. 19-22. 25. The cell of claim 24, wherein the cell is an effector cell. 26. The cell of claim 25, wherein the effector cell is a T cell. 27. A method of treating a medical condition characterized by CD19 expression in an object, comprising the step of administering to said object an antibody or antigen-binding fragment according to any one of paragraphs. 1-16, a bispecific antibody according to claim 17 or 18, a chimeric antigen receptor according to any one of claims. 19-22, nucleic acid molecule according to claim 23, effector cell according to claim 25 or 26. 28. The method of claim 27, wherein the medical condition characterized by CD19 expression is rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), diabetes, or scleroderma. 29. The method of claim 27, wherein the medical condition characterized by CD19 expression is B-cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt's lymphoma, non-Hodgkin's lymphoma, or acute myeloid leukemia. 30. The method of claim 27 wherein the medical condition characterized by CD19 expression is cancer. 31. A method for inhibiting tumor growth or killing tumor cells, wherein the tumor expresses CD19, the method comprising the step of contact of tumor cells with an antibody or antigen-binding fragment according to any one of paragraphs. 1-16, a bispecific antibody according to claim 17 or 18, a chimeric antigen receptor according to any one of paragraphs. 19-22, a nucleic acid molecule according to claim 23 or an effector cell according to claim 25 or 26, wherein the contact is made under conditions and for a time sufficient to inhibit tumor growth or kill tumor cells. 32. A method for controlling T cells to kill target cells expressing CD19, the method comprising the step of contact of one or more target cells expressing CD19 with one or more T cells and an antibody or antigen-binding fragment according to any one of paragraphs. 1-16 or a bispecific antibody according to claim 17 or 18 or contacting one or more target cells expressing CD19 with an effector cell according to claim 25 or 26, where the effector cell is a T cell, wherein the contact is made under conditions and for a time sufficient for the T cells to which the antibody or antigen-binding fragment or bispecific antibody binds to mediate the killing of the target cells, or for the effector cell to mediate the killing of the target cells.

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