Anti-HIV antibody 10-1074 variant

Abstract

The present disclosure provides optimized broadly neutralizing anti-HIV antibodies having modified light chain variable regions and / or heavy chain variable regions that result in improved biophysical characteristics. The present disclosure also provides methods of producing these anti-HIV antibodies and methods of using the same.

Description

Anti-HIV antibody 10-1074 variant

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62 / 731,356, filed on September 14, 2018. The above application is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grant No. P01 AI081677 awarded by the NIH. The government has certain rights in this invention. Technical Field Field of the Invention

[0005] The present invention relates generally to broad and potent antibodies against human immunodeficiency virus ("HIV"), and more particularly to anti-HIV antibody 10-1074 variants and uses thereof. Background Art

[0006]

[0007] HIV causes acquired immunodeficiency syndrome (AIDS), a condition characterized in humans by severe immunosuppression and clinical features of malignant tumors, including wasting syndrome, degeneration of the central nervous system, and life-threatening opportunistic infections. Since its discovery in 1981, HIV type 1 (HIV-1) has caused at least 25 million deaths worldwide. It is predicted that even if the HIV infection rate drops by 2.5% per year, 20 to 60 million people will be infected in the next 20 years. There is a need for therapeutic agents and methods for treating or suppressing HIV infection.

[0008] Some individuals infected with HIV show broadly neutralizing IgG antibodies in their serum. Although these antibodies are potentially important for designing effective vaccines, little is known about their specificity and activity. In animal models, passive transfer of neutralizing antibodies can help protect against viral attack. Neutralizing antibody responses can also be developed in individuals infected with HIV, but the detailed composition of serum responses has not yet been fully revealed. Summary of the Invention

[0009] The present disclosure relates to a new class of broadly neutralizing anti-HIV antibodies having modified light chain variable regions and / or heavy chain variable regions resulting in improved biophysical properties, and methods of producing and using the same.

[0010] Thus, in a first aspect, the present disclosure provides an isolated anti-HIV antibody, or antigen-binding portion thereof, comprising a light chain variable region having a light chain amino acid sequence that is at least 75% identical to a polypeptide sequence selected from the group consisting of the light chain variable regions of SEQ ID NOs: 3-13, 22, 24-28, 35-39, 43-45, and 47. The isolated anti-HIV antibody, or antigen-binding portion thereof, comprises one or more light chain substitutions at one or more residues within or outside the light chain variable region. The one or more residues are selected from the group consisting of: LmdV:Y2, LmdV:R7, LmdV:P9, LmdV:E17, LmdV:H46, LmdV:P81.1, LmdV:I81.3, LmdV:N82, LmdV:R88, LmdV:D110, and LmdV:A142.

[0011] In another aspect, the present disclosure provides an isolated anti-HIV antibody, or antigen-binding portion thereof, comprising a heavy chain variable region having a heavy chain amino acid sequence that is at least 75% identical to a polypeptide sequence selected from the group consisting of the heavy chain variable regions of SEQ ID NOs: 61-94. The isolated anti-HIV antibody, or antigen-binding portion thereof, comprises one or more heavy chain substitutions at one or more residues within or outside the heavy chain variable region. The one or more residues are selected from the group consisting of: HV:D29, HV:S47, HV:N75, HV:V79, HV:R82, HV:L89, HV:T108, and HV:K141.

[0012] In another aspect, the present disclosure provides an isolated anti-HIV antibody, or antigen-binding portion thereof, comprising a light chain variable region having a light chain amino acid sequence that is at least 75% identical to a polypeptide sequence selected from the group consisting of the light chain variable regions of SEQ ID NOs: 3-13, 22, 24-28, 35-39, 43-45, and 47. The isolated anti-HIV antibody, or antigen-binding portion thereof, comprises one or more light chain substitutions at one or more residues selected from the group consisting of LmdV:Y2, LmdV:R7, LmdV:P9, LmdV:E17, LmdV:H46, LmdV:P81.1, LmdV:I81.3, LmdV:N82, LmdV:R88, LmdV:D110, and LmdV:A142. The anti-HIV antibody, or antigen-binding portion thereof, further comprises a heavy chain variable region having a heavy chain amino acid sequence that is at least 75% identical to a polypeptide sequence selected from the group consisting of the heavy chain variable regions of SEQ ID NOs: 61-94. The isolated anti-HIV antibody, or antigen-binding portion thereof, comprises one or more heavy chain substitutions at one or more residues selected from the group consisting of: HV:D29, HV:S47, HV:N75, HV:V79, HV:R82, HV:L89, HV:T108, and HV:K141.

[0013] In some embodiments, the isolated anti-HIV antibody, or antigen-binding portion thereof, comprises one or more light chain substitutions selected from the group consisting of LmdV:Y2P, LmdV:R7P, LmdV:P9S, LmdV:E17Q, LmdV:H46Q, LmdV:P81.1N, LmdV:I81.3S, LmdV:N82G, LmdV:R88T, LmdV:D110E, and LmdV:A142G, or conservative substitutions thereof (i.e., LmdV:P9C, LmdV:P9T, LmdV:E17N, LmdV:H46N, LmdV:P81.1Q, LmdV:R88C, LmdV:R88S).

[0014] In some embodiments, the isolated anti-HIV antibody, or antigen-binding portion thereof, comprises one or more heavy chain substitutions selected from the group consisting of: HV:D29G, HV:S47P, HV:N75Q, HV:V79T, HV:R82V, HV:L89F, HV:T108R, and HV:K141Q, or conservative substitutions thereof (i.e., HV:L89W, HV:L89Y, HV:T108H, HV:T108K, HV:K141N).

[0015] In some embodiments, the isolated anti-HIV antibody or antigen-binding portion thereof comprises one or more light chain substitutions selected from the group consisting of: LmdV:Y2P, LmdV:R7P, LmdV:P9S, LmdV:E17Q, LmdV:H46Q, LmdV:P81.1N, LmdV:I81.3S, LmdV:N82G, LmdV:R88T, LmdV:D110E, and LmdV:A142G, or conservative substitutions thereof (i.e., LmdV:P9C, LmdV:P9T, LmdV:H46Q, LmdV:P81.1N, LmdV:I81.3S, LmdV:N82G, LmdV:R88T, LmdV:D110E, and LmdV:A142G). V:E17N, LmdV:H46N, LmdV:P81.1Q, LmdV:R88C, LmdV:R88S), and one or more heavy chain substitutions selected from the group consisting of: HV:D29G, HV:S47P, HV:N75Q, HV:V79T, HV:R82V, HV:L89F, HV:T108R and HV:K141Q or conservative substitutions thereof (i.e., HV:L89W, HV:L89Y, HV:T108H, HV:T108K, HV:K141N).

[0016] In some embodiments, the light chain amino acid sequence is at least 75% identical to the light chain variable region of SEQ ID NO.: 3 and comprises a LmdV:Y2P substitution or a conservative substitution of proline at LmdV:Y2.

[0017] In some embodiments, the heavy chain amino acid sequence is at least 75% identical to the heavy chain variable region of SEQ ID NO.: 63 and comprises a HV:V79T substitution or a conservative substitution of threonine at HV:V79.

[0018] In some embodiments, the heavy chain amino acid sequence is at least 75% identical to the heavy chain variable region of SEQ ID NO.: 64 and comprises a HV:R82V substitution or a conservative substitution of valine at HV:R82.

[0019] In some embodiments, the heavy chain amino acid sequence is at least 75% identical to the heavy chain variable region of SEQ ID NO.: 65 and comprises a HV:L89F substitution or a conservative substitution of phenylalanine at HV:L89.

[0020] In some embodiments, the heavy chain amino acid sequence is at least 75% identical to the heavy chain variable region of SEQ ID NO.: 66 and comprises a HV:T108R substitution or a conservative substitution of arginine at HV:T108.

[0021] In some embodiments, the light chain amino acid sequence is at least 75% identical to the light chain variable region of SEQ ID NO.: 22 and comprises a LmdV:Y2P substitution or a conservative substitution of proline at LmdV:Y2, and the heavy chain amino acid sequence is at least 75% identical to the heavy chain variable region of SEQ ID NO.: 69 and comprises a HV:R82V substitution or a conservative substitution of valine at HV:R82, and a HV:T108R substitution or a conservative substitution of arginine at HV:T108.

[0022] In some embodiments, the heavy chain amino acid sequence is at least 75% identical to the heavy chain variable region of SEQ ID NO.: 70 and comprises a HV:V79T substitution or a conservative substitution of threonine at HV:V79, a HV:L89F substitution or a conservative substitution of phenylalanine at HV:L89, a HV:T108R substitution or a conservative substitution of arginine at HV:T108.

[0023] In some embodiments, the light chain amino acid sequence is at least 75% identical to the light chain variable region of SEQ ID NO.: 24 and comprises a LmdV:Y2P substitution or a conservative substitution of proline at LmdV:Y2, and the heavy chain amino acid sequence is at least 75% identical to the heavy chain variable region of SEQ ID NO.: 71 and comprises a HV:V79T substitution or a conservative substitution of threonine at HV:V79, a HV:L89F substitution or a conservative substitution of phenylalanine at HV:L89, a HV:T108R substitution or a conservative substitution of arginine at HV:T108.

[0024] In some embodiments, the isolated anti-HIV antibody, or antigen-binding portion thereof, comprises SEQ ID NO.: 3. In some embodiments, the isolated anti-HIV antibody, or antigen-binding portion thereof, comprises SEQ NO.: 63, 64, 65, 66, or 70. In some embodiments, the light chain variable region comprises the light chain variable region of SEQ NO.: 22, and the heavy chain variable region comprises the heavy chain variable region of SEQ NO.: 69. In some embodiments, the light chain variable region comprises the light chain variable region of SEQ NO.: 24, and the heavy chain variable region comprises the heavy chain variable region of SEQ NO.: 71.

[0025] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the above-mentioned anti-HIV antibody or antigen-binding portion and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is an anti-HIV-1 broadly neutralizing antibody, such as 3BNC117.

[0026] In another aspect, the present disclosure further provides nucleic acids or codon-optimized nucleic acids encoding the above-mentioned anti-HIV antibodies or antigen-binding portions thereof, vectors or vector systems having at least one of the above-mentioned nucleic acids, and cells having at least one of the above-mentioned nucleic acids.

[0027] In another aspect, the present disclosure provides a method for preparing a recombinant anti-HIV antibody or antigen-binding portion thereof, comprising obtaining the cultured cells described above, culturing the cells in a culture medium under conditions that allow expression of a polypeptide encoded by the vector and assembly of the antibody or fragment thereof, and purifying the antibody or fragment from the cultured cells or cell culture medium.

[0028] In another aspect, the present disclosure provides a method for preventing or treating HIV infection or HIV-related diseases. The method includes identifying a patient in need of such prevention or treatment, and administering to the patient a first therapeutic agent having a therapeutically effective amount of at least one of the above-mentioned anti-HIV antibodies or antigen-binding portions thereof. The method may further include administering a second therapeutic agent. The second therapeutic agent may be administered before, simultaneously with, or after the administration of the anti-HIV antibody or antigen-binding portion thereof. In some embodiments, the second therapeutic agent is an anti-HIV-1 broadly neutralizing antibody, such as 3BNC117.

[0029] In another aspect, the present disclosure further provides a kit comprising a pharmaceutically acceptable dosage unit of at least one of the above-described isolated anti-HIV antibodies or antigen-binding portions thereof in a pharmaceutically effective amount. The kit may further comprise a pharmaceutically acceptable dosage unit of an anti-HIV agent in a pharmaceutically effective amount. The two pharmaceutically acceptable dosage units may optionally be in the form of a single pharmaceutically acceptable dosage unit. Exemplary anti-HIV agents may be selected from the group consisting of non-nucleoside reverse transcriptase inhibitors, protease inhibitors, entry or fusion inhibitors, and integrase inhibitors. In some embodiments, the anti-HIV agent is an anti-HIV broadly neutralizing antibody, such as 3BNC117.

[0030] The foregoing summary of the invention is not intended to define every aspect of the present disclosure, and other aspects are described in other parts of the detailed description, for example, below. The entire document is intended to be associated as a unified disclosure, and it should be understood that all combinations of features described herein are considered, even if the combination of features does not appear together in the same sentence, paragraph or section of the document. Other features and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples briefly set forth specific embodiments of the present disclosure, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will be apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figures 1A and 1B (collectively referred to as "Figure 1") show the characterization of anti-HIV antibody 10-1074 variants MS-194 (Figure 1A) and MS-203 (Figure 1B) by high performance size exclusion chromatography ("HP-SEC") before and after virus neutralization. Arrows indicate peaks in the HP-SEC profiles corresponding to oligomeric species formed during virus inactivation.

[0033] Figure 2 shows quantification of the extent of aggregation as represented by the levels of high molecular weight ("HMW") and oligomers after each purification step of the 10-1074 antibody variants MS-194, MS-200, MS-201, and MS-203.

[0034] Figure 3 shows the HMW levels of 10-1074 antibody variants MS-194, MS-200, MS-201 and MS-203 during incubation at 40°C for up to 13 weeks.

[0035] Figure 4 shows the levels of sub-visible particle formation by the 10-1074 antibody variants MS-194, MS-200, MS-201 and MS-203 over 6 and 13 weeks.

[0036] Figures 5A, 5B, and 5C (collectively referred to as "Figure 5") show delayed viral rebound during analytical treatment interruption (ATI) using a combination of 3BNC117 and 10-1074. Figure 5A shows the study design of a Phase 1b clinical trial in which a combination of 3BNC117 and 10-1074 (two potent monoclonal anti-HIV-1 broadly neutralizing antibodies targeting independent sites on the HIV-1 envelope spike) was administered during the ATI. Red and blue triangles represent 3BNC117 and 10-1074 infusions, respectively. Figure 5B shows plasma HIV-1 RNA levels (black; left y-axis) and bNAb serum concentrations (3BNC117, red; 10-1074, blue; right y-axis) in 9 participants (left) who were sensitive to bNAbs and 2 participants (right) who had pre-existing resistance to one of the antibodies. Red and blue triangles represent infusions of 3BNC117 and 10-1074, respectively. Serum antibody concentrations were determined by the TZM-b1 assay. The gray shaded area represents the time on ART. The lower limit of detection for HIV-1 RNA is 20 copies / ml. Figure 5C shows a Kaplan-Meier plot summarizing the time to viral rebound in participants with HIV-1 RNA <20 copies / ml 2 weeks before and at the start of ATI (n=11, left), participants who were sensitive to both antibodies (n=9, center), and participants who showed pre-existing resistance to one of the antibodies (n=2, right). The Y-axis represents the percentage of participants who maintained viral suppression. The X-axis represents the number of weeks after the start of ATI. The blue line represents participants who received the combination of 3BNC117+10-1074. The red dotted line represents a group of individuals who received only 3BNC117 during the ATI (n=13), and the black dotted line represents a group of participants who underwent ATI without intervention (n=52).

[0037] Figures 6A, 6B, 6C, 6D, 6E, and 6F (collectively referred to as "Figure 6") show demographic data, CD4 + T cells and pharamacokinetics of 3BNC117 and 10-1074. The table in Figure 6A shows baseline participant demographics. *NNRTI - non-nucleoside reverse transcriptase inhibitor. Figure 6B shows absolute CD4 T cells at screening (n=15), day 0 (n=15), viral rebound (n=13), and end of study (n=15). + T cell count and CD3 + CD4 in T cells +T cell percentages (see also Supplementary Table 2). The last available time point after resuppression was used as the endpoint of the study time point for participants who restarted ART. The red line represents the mean, and the error bars represent the standard deviation. Two-tailed paired t-tests were used to compare CD4 between day 0 and the viral rebound time point. + T cell counts were used to obtain P values. Figures 6C and 6D show the levels of 3BNC117 (red) and 10-1074 (blue) in serum (n=15) determined by the TZM-bl assay (Figure 6C) and ELISA (Figure 6D). The curve represents the mean serum antibody concentration, and the error bars represent the standard deviation. The red and blue triangles represent the infusions of 3BNC117 and 10-1074, respectively. In the TZM-bl assay, the detection limits of 3BNC117 and 10-1074 were 0.46 μg / ml and 0.01 μg / ml, respectively (Figure 6C). In the ELISA, the detection limits were 0.78 μg / ml and 0.41 μg / ml, respectively (Figure 6D). In the case where participants received only 2 infusions (9245, 9249, and 9253) due to early viral rebound, only the antibody concentration up to the second infusion was included. The half-life of each bNAb is expressed in days. Figures 6E and 6F show the half-lives of the two antibodies measured by TZM-b1 assay (Figure 6E) and ELISA (Figure 6F). Each point represents a single participant. The half-lives of the two antibodies from the 15 participants in the study are shown. The black line represents the mean and standard deviation (n=15). P values ​​were obtained by comparing the two antibodies using a two-tailed unpaired t-test.

[0038] Figures 7A, 7B, and 7C (collectively, "Figure 7") show amino acid variants at the 10-1074 contact site and bNAb sensitivity of reactivated latent and rebound virus. The set of graphs in Figure 7A shows glycans at the Env contact site of 10-1074 at the G(D / N)IR motif (positions 324-327 according to HXB2 numbering) and at the potential N-linked glycosylation site at position 332 (NxS / T motif at positions 332-334). The figure shows 7 participants who rebounded before week 30 who were sensitive to bNAbs (left) and 2 individuals with pre-existing resistance to one of the two antibodies (right). LR indicates resistance to reactivation by Q 2VOA separates latent pool (latent reservoir) virus, and RB represents rebound virus separated by SGA (plasma) or viral outgrowth (viral outgrowth) (PBMC). Each amino acid is represented by a color, and the frequency of each amino acid is represented by the height of the rectangle. The shaded rectangle represents that there is no change at the indicated position between the latent pool virus and the rebound virus. The full-color rectangle represents that there is an amino acid residue that changes in distribution between the pool virus and the rebound virus. The dot plots of Figures 7B and 7C show the IC80 (μg / ml) of 3BNC117 (Figure 7B, left figure) and 10-1074 (Figure 7C, right figure) for latent virus and rebound virus determined by TZM-b1 neutralization assay. Q from week 2 and week 12 2 Latent viruses derived from VOA are shown as black and gray circles, respectively. For rebound viruses derived from outgrowth cultures, the highest IC80 determined is shown as a red circle. For 9246, 9252, 9245, and 9251, viruses could not be obtained from rebound outgrowth cultures, and pseudoviruses were generated from Q 2 The env sequences of VOA and plasma SGA were prepared.

[0039] Figure 8 shows a comparison of circulating latent pool virus and rebound virus. Q from 3 of the 7 participants (9242, 9243, and 9252) who rebounded before week 30 2 Maximum likelihood phylogenetic tree of full-length env sequences of viruses isolated from VOA, rebound plasma SGA, and rebound PBMC growth cultures. Open and closed black rectangles represent Q from weeks 2 and 12, respectively. 2 Viruses of VOA origin. Viruses obtained during rebound are represented by red rectangles (plasma SGA) and red asterisks (rebound PBMC growth cultures). Asterisks indicate nodes with significant bootstrap values ​​(bootstrap support ≥ 70%). Boxes indicate, where possible, the IC80 (μg / ml) of 3BNC117 and 10-1074 for representative viruses of the entire phylogenetic tree and clones. Asterisks in boxes indicate IC100 values ​​> 50 μg / ml.

[0040] Figures 9A and 9B (collectively referred to as "Figure 9") show the distribution of circulating latent pool viruses and rebound viruses. Figure 9A is a set of Venn diagrams showing the distribution of Q from week 2 (blue) and week 12 (grey) 2Sequence identity between env sequences obtained from VOA, plasma SGA, or rebound PBMC growth cultures (red) during viral rebound. The area of ​​overlap is proportional to the number of identical sequences. The number of sequences obtained is shown. Figure 9B shows the sequence identity between env sequences obtained by Q 2 CD4 at 2 and 12 weeks as determined by VOA + Infectious units per million (IUPM) of T cells. Participants with an IUPM above and below 0.1 are shown at the top and bottom, respectively. There was no statistical difference between the two time points (P = 0.078 (paired t-test)). DETAILED DESCRIPTION

[0041]

[0042] The present disclosure is based, at least in part, on the unexpected discovery of a new class of broadly neutralizing antibodies (bNAbs) against HIV that recognize carbohydrate-dependent epitopes on gp120, including complex N-glycans.

[0043] Antibodies are crucial for the success of most vaccines, and antibodies against HIV appear to be the only correlate of protection in the current RV144 anti-HIV vaccine trial. Some HIV-1 infected patients develop broadly neutralizing serological activity against the gp160 viral spike 2 to 4 years after infection, but these antibodies generally do not protect infected humans because autologous viruses escape through mutations. Nevertheless, broadly neutralizing activity puts selective pressure on the virus, and passive transfer of broadly neutralizing antibodies (bNAbs) to macaques can protect them from SHIV infection. Therefore, it has been proposed that vaccines that induce such antibodies could protect humans from HIV infection.

[0044] The development of single-cell antibody cloning technology has shown that bNAbs target multiple different epitopes on the HIV-1 gp160 spike. The most potent HIV-1 bNAbs recognize the CD4 binding site (CD4bs) (Science 333(6049):1633-1637; Nature 477(7365):466-470; Science 334(6060):1289-1293) and carbohydrate-dependent epitopes associated with the variable loops (Nature 477(7365):466-470; Science 326(5950):285-289; Science 334(6059):1097-1103; Nature 480(7377):336-343), including the V1 / V2 (PG9 / PG16) (Science 326(5950):285-289) and V3 loop (PGT) (Nature 477(7365):466-470). Little is known about carbohydrate-dependent epitopes, as the antibodies studied to date are either unique examples or members of small clonal families.

[0045] To better understand the neutralizing antibody response to HIV-1 and the epitopes targeted by PGT antibodies, members of a large clonal family that dominated the gp160-specific IgG memory response from patients infected with clade A that produced PGT121 have been isolated. The isolation of PGT121 is described in more detail in PCT / US13 / 65696. Based on sequence, binding affinity, neutralizing activity, and recognition of carbohydrates and the V3 loop, PGT121 antibodies can be divided into two groups: PGT121-like and 10-1074-like groups. 10-1074 and related family members exhibit unusually potent neutralization, including broad reactivity against newly transmitted viruses. Unlike previously characterized carbohydrate-dependent bNAbs, PGT121 binds to complex rather than high-mannose N-glycans in glycan microarray experiments. The 10-1074 group showed significant efficacy and breadth, although no binding to apoprotein glycans was detected. The crystal structures of unliganded PGT121, 10-1074, and their germline precursors revealed that distinct carbohydrate recognition is localized to the cleft between CDRH2 and CDRH3 that is occupied by complex N-glycans in the PGT121 structure alone. The exchanged glycan contact residues between PGT121 and 10-1074 confirm the importance of these residues in neutralizing activity.

[0046] Because the biophysical stability of monoclonal antibodies is an important determinant of their usefulness and commercial value, the present disclosure proposes a method for optimizing the biophysical characteristics of 10-1074 broadly neutralizing antibodies. For example, a series of substitutions were performed to identify potential unstable residues in the Fv region of 10-1074 broadly neutralizing antibodies. These residues may, alone or in combination, lead to instability at low pH, increase sensitivity to chemical degradation, or lead to aggregation during production or long-term storage. Based on this analysis, a series of variants were designed to optimize the desired characteristics using a combinatorial residue replacement technique while maintaining efficacy. The optimization process is divided into different stages, the first stage being to identify single residues in the framework region that may cause instability. Specifically, anti-HIV 10-1074 antibody variants (as shown in Tables 2-7 and 9) were produced by transient expression, each of which contained a single residue modification of the identified amino acids. As shown in Tables 8-16, the retention of the neutralizing activity of the variants and the desired biophysical characteristics were characterized. Five different amino acid residues were identified that showed an increase in the desired biophysical characteristics and did not affect neutralization: LmdV: Y2, HV: V79, HV: R82, HV: L89, and HV: T108. The residues were used to generate a variant library containing all possible combinations of the five amino acids (as shown in Tables 2-7 and 12). Variants were again produced by transient expression, and the purified combinatorial variants were analyzed for retention of neutralizing activity and the desired biophysical characteristics. Three variants, MS-200, MS-201, and MS-202, were identified from the combinatorial library for a more in-depth analysis including expression, purification, and storage stability to identify combinatorial variants with optimized characteristics including increased thermal stability, increased resistance to chemical unfolding, increased solubility, and increased resistance to aggregation during storage.

[0047] Isolated anti-HIV antibodies, pharmaceutical compositions and kits

[0048] Thus, in one aspect, the present disclosure provides an isolated anti-HIV antibody, or antigen-binding portion thereof, comprising a light chain variable region having a light chain amino acid sequence that is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to a polypeptide sequence selected from the group consisting of the light chain variable regions of SEQ ID NOs: 3-13, 22, 24-28, 35-39, 43-45, and 47 (Table 2). The isolated anti-HIV antibody, or antigen-binding portion thereof, may comprise one or more light chain substitutions at one or more residues within or outside the light chain variable region. The substituted residues may be one or more of the following: LmdV:Y2, LmdV:R7, LmdV:P9, LmdV:E17, LmdV:H46, LmdV:P81.1, LmdV:I81.3, LmdV:N82, LmdV:R88, LmdV:D110, and LmdV:A142.

[0049] Also provided are isolated anti-HIV antibodies, or antigen-binding portions thereof, comprising a heavy chain variable region having a heavy chain amino acid sequence that is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to a polypeptide sequence selected from the group consisting of the heavy chain variable regions of SEQ ID NOs: 61-94 (Table 3). The isolated anti-HIV antibodies, or antigen-binding portions thereof, comprise one or more heavy chain substitutions at one or more residues within or outside the heavy chain variable region. The substituted residues may be one or more of: HV:D29, HV:S47, HV:N75, HV:V79, HV:R82, HV:L89, HV:T108, and HV:K141.

[0050] In another aspect, the present disclosure provides an isolated anti-HIV antibody, or antigen-binding portion thereof, comprising a light chain variable region having a light chain amino acid sequence that is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to a polypeptide sequence selected from the group consisting of the light chain variable regions of SEQ ID NOs: 3-13, 22, 24-28, 35-39, 43-45, and 47 (Table 2). The isolated anti-HIV antibody, or antigen-binding portion thereof, comprises one or more light chain substitutions at one or more of the following residues: LmdV:Y2, LmdV:R7, LmdV:P9, LmdV:E17, LmdV:H46, LmdV:P81.1, LmdV:I81.3, LmdV:N82, LmdV:R88, LmdV:D110, and LmdV:A142. The anti-HIV antibody, or antigen-binding portion thereof, further comprises a heavy chain variable region having a heavy chain amino acid sequence that is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to a polypeptide sequence selected from the group consisting of the heavy chain variable regions of SEQ ID NOs: 61-94 (Table 3). The isolated anti-HIV antibody, or antigen-binding portion thereof, comprises one or more heavy chain substitutions at one or more of the following residues: HV:D29, HV:S47, HV:N75, HV:V79, HV:R82, HV:L89, HV:T108, and HV:K141.

[0051] In some embodiments, the isolated anti-HIV antibody or antigen-binding portion thereof comprises one or more of the following light chain substitutions: LmdV:Y2P, LmdV:R7P, LmdV:P9S, LmdV:E17Q, LmdV:H46Q, LmdV:P81.1N, LmdV:I81.3S, LmdV:N82G, LmdV:R88T, LmdV:D110E and LmdV:A142G or conservative substitutions thereof (i.e., LmdV:P9C, LmdV:P9T, LmdV:E17N, LmdV:H46N, LmdV:P81.1Q, LmdV:R88C, LmdV:R88S).

[0052] In some embodiments, the isolated anti-HIV antibody or antigen-binding portion thereof comprises one or more of the following heavy chain substitutions: HV:D29G, HV:S47P, HV:N75Q, HV:V79T, HV:R82V, HV:L89F, HV:T108R and HV:K141Q or conservative substitutions thereof (i.e., HV:L89W, HV:L89Y, HV:T108H, HV:T108K, HV:K141N).

[0053] In some embodiments, the isolated anti-HIV antibody or antigen-binding portion thereof comprises one or more of the following light chain substitutions: LmdV:Y2P, LmdV:R7P, LmdV:P9S, LmdV:E17Q, LmdV:H46Q, LmdV:P81.1N, LmdV:I81.3S, LmdV:N82G, LmdV:R88T, LmdV:D110E, and LmdV:A142G, or conservative substitutions thereof (i.e., LmdV:P9C, LmdV:P9T, LmdV: V:E17N, LmdV:H46N, LmdV:P81.1Q, LmdV:R88C, LmdV:R88S), and one or more of the following heavy chain substitutions: HV:D29G, HV:S47P, HV:N75Q, HV:V79T, HV:R82V, HV:L89F, HV:T108R and HV:K141Q or conservative substitutions thereof (i.e., HV:L89W, HV:L89Y, HV:T108H, HV:T108K, HV:K141N).

[0054] In some embodiments, the light chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the light chain variable region of SEQ ID NO.: 3 and comprises a LmdV:Y2P substitution or a conservative substitution of proline at LmdV:Y2.

[0055] In some embodiments, the heavy chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the heavy chain variable region of SEQ ID NO.: 63 and comprises a HV:V79T substitution or a conservative substitution of threonine at HV:V79.

[0056] In some embodiments, the heavy chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the heavy chain variable region of SEQ ID NO.: 64 and comprises a HV:R82V substitution or a conservative substitution of valine at HV:R82.

[0057] In some embodiments, the heavy chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the heavy chain variable region of SEQ ID NO.: 65 and comprises a HV:L89F substitution or a conservative substitution of phenylalanine at HV:L89.

[0058] In some embodiments, the heavy chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the heavy chain variable region of SEQ ID NO.: 66 and comprises a HV:T108R substitution or a conservative substitution of arginine at HV:T108.

[0059] In some embodiments, the light chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the light chain variable region of SEQ ID NO.: 22 and comprises a LmdV: Y2P substitution or a conservative substitution of proline at LmdV: Y2, and the heavy chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the heavy chain variable region of SEQ ID NO.: 69 and comprises a HV: R82V substitution or a conservative substitution of valine at HV: R82, and a HV: T108R substitution or a conservative substitution of arginine at HV: T108.

[0060] In some embodiments, the heavy chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the heavy chain variable region of SEQ ID NO.: 70 and comprises a HV:V79T substitution or a conservative substitution of threonine at HV:V79, a HV:L89F substitution or a conservative substitution of phenylalanine at HV:L89, and a HV:T108R substitution or a conservative substitution of arginine at HV:T108.

[0061] In some embodiments, the light chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the light chain variable region of SEQ ID NO.: 24 and comprises a LmdV:Y2P substitution or a conservative substitution of proline at LmdV:Y2, and the heavy chain amino acid sequence is at least 75% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%) identical to the heavy chain variable region of SEQ SEQ ID NO.: 71 and comprises a HV:V79T substitution or a conservative substitution of threonine at HV:V79, a HV:L89F substitution or a conservative substitution of phenylalanine at HV:L89, and a HV:T108R substitution or a conservative substitution of arginine at HV:T108.

[0062] In some embodiments, the isolated anti-HIV antibody, or antigen-binding portion thereof, comprises SEQ ID NO.: 3. In some embodiments, the isolated anti-HIV antibody, or antigen-binding portion thereof, comprises SEQ NO.: 63, 64, 65, 66, or 70. In some embodiments, the light chain variable region comprises the light chain variable region of SEQ NO.: 22, and the heavy chain variable region comprises the heavy chain variable region of SEQ NO.: 69. In some embodiments, the light chain variable region comprises the light chain variable region of SEQ NO.: 24, and the heavy chain variable region comprises the heavy chain variable region of SEQ NO.: 71.

[0063] The positions of the variable domain residues are numbered according to the structure-based numbering system of AHo (Honegger, A., & Plückthun, A. (2001). Journal of Molecular Biology, 309 (3), 657-70.). Table 1 shows exemplary residue numbers for the variable domains of MS-194. The abbreviations used in Table 1 are described as follows. "Ldr" refers to the leader sequence (e.g., AKA signal sequence or signal peptide). "Mat.Linear" refers to the linear number of the mature form of the protein chain. "LmdV" refers to the variable region in the lambda type light chain.

[0064] As used herein, the term "antibody" (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies) and antibody fragments. Therefore, the term "antibody" as used in any context within this specification is intended to include, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant fragments or their specific binding members, including but not limited to Fab, F(ab')2, Fv and scFv (single chain or related entities). It should be understood in the art that antibodies are glycoproteins or antigen binding portions thereof having at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2 and CH3). The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable regions of both heavy and light chains consist of framework regions (FWRs) and complementarity-determining regions (CDRs). The four FWRs are relatively conserved, while the CDRs (CDR1, CDR2, and CDR3) represent the hypervariable regions and are arranged from the NH2-terminus to the COOH-terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The variable regions of both heavy and light chains contain the binding domain that interacts with the antigen, while the constant regions, depending on the isotype, mediate the binding of the immunoglobulin to host tissues or factors.

[0065] As used herein, also included within the definition of "antibody" are chimeric, humanized, and recombinant antibodies, human antibodies produced from transgenic non-human animals, and antibodies selected from libraries using enrichment techniques available to the skilled artisan.

[0066] The term "variable" refers to the fact that certain segments of the variable (V) domain differ greatly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the amino acid span of the variable region. Instead, the V region consists of relatively unchanging stretches of 15-30 amino acids called framework regions (FRs), separated by shorter regions of extremely variable length, called "hypervariable regions," which can be 9-12 amino acids in length. The variable regions of native heavy and light chains each contain four FRs, primarily adopting a β-sheet configuration, and are connected by three hypervariable regions that form loops that connect and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are tightly bound together by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antibody's antigen-binding site (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0067] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from a "complementarity determining region" ("CDR").

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The term "polyclonal antibody" refers to a preparation that includes different antibodies directed against different determinants ("epitopes").

[0069] The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). The invention provides variable region antigen-binding sequences derived from human antibodies. Thus, the chimeric antibodies of primary interest herein include antibodies having one or more human antigen-binding sequences (e.g., CDRs) and containing one or more sequences derived from non-human antibodies (e.g., FR or C region sequences). In addition, the chimeric antibodies included herein are those comprising a human variable region antigen-binding sequence of one antibody class or subclass and another sequence (e.g., FR or C region sequence) derived from another antibody class or subclass.

[0070] It is generally considered that "humanized antibodies" are human antibodies with one or more amino acid residues of non-human origin introduced. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable region. Humanization can be performed according to the method of Winter and colleagues (see, e.g., Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) by replacing the corresponding sequences of a human antibody with the sequences of the imported hypervariable regions. Thus, such "humanized" antibodies are chimeric antibodies (see, e.g., U.S. Patent No. 4,816,567), in which substantially less than the entire human variable region has been substituted with the corresponding sequence from a non-human species.

[0071] "Antibody fragments" comprise a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see, e.g., U.S. Patent No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0072] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This fragment contains a dimer of a heavy chain and a light chain variable region domain in tight non-covalent association. From the folding of these two domains, six hypervariable regions (three loops each for the H and L chains) are generated, which contribute the amino acid residues for antigen binding and give the antibody antigen binding specificity. However, even a single variable region (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the complete binding site.

[0073] "Single-chain Fv" ("sFv" or "scFv") is an antibody fragment comprising the VH and VL antibody domains linked to a single polypeptide chain. The sFv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the sFv to form the desired antigen-binding structure. For a review of sFv, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

[0074] The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments with a short linker (about 5-10 residues) between the VH and VL domains, thereby achieving interchain pairing of the V domains rather than intrachain pairing, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0075] Domain antibodies (dAbs) that can be produced in fully human form are the minimum antigen-binding fragments of known antibodies, ranging between approximately 11kDa and 15kDa. DAbs are the robust variable regions of immunoglobulin heavy and light chains (VH and VL, respectively). They are highly expressed in microbial cell cultures, demonstrate favorable biophysical properties, including, for example, but not limited to, solubility and temperature stability, and are very suitable for selection and affinity maturation by, for example, phage display, an in vitro selection system. DAbs have biological activity as monomers, and due to their small size and inherent stability, can be designed into larger molecules to create drugs with prolonged serum half-life or other pharmacological activities. The example of this technology has been described in, for example, following: in WO9425591, antibodies derived from Camelidae heavy chain Ig have been described, and in US20030130496, single domain fully human antibodies have been described for separation from phage libraries.

[0076] Fv and sFv are the only species with complete binding sites that lack constant regions. Therefore, they are suitable for reducing nonspecific binding during in vivo use. sFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl termini of sFv. See, for example, Antibody Engineering, ed. Borrebaeck, supra. Antibody fragments can also be "linear antibodies," such as those described in U.S. Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0077] In certain embodiments, the antibody of the invention is bispecific or multispecific. Bispecific antibodies are antibodies with binding specificity to at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of a single antigen. Other such antibodies can bind to a first antigen binding site and a binding site for a second antigen. Alternatively, the anti-HIV arm can be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD3) or an Fc receptor (FcγR) of IgG, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), thereby concentrating and localizing the cellular defense mechanism to infected cells. Bispecific antibodies can also be used to localize cytotoxic agents to infected cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). For example, WO 96 / 16673 describes bispecific anti-ErbB2 / anti-FcγRIII antibodies, and U.S. Pat. No. 5,837,234 discloses bispecific anti-ErbB2 / anti-FcγRI antibodies. For example, bispecific anti-ErbB2 / Fcα antibodies are reported in WO 98 / 02463; U.S. Pat. No. 5,821,337 teaches bispecific anti-ErbB2 / anti-CD3 antibodies. See also, for example, Mouquet et al., “Polyreactivity Increases The Apparent Affinity Of Anti-HIV Antibodies By Heteroligation.” Nature. 467, 591-5 (2010), and Mouquet et al., “Enhanced HIV-1 neutralization by antibody heteroligation.” Proc Natl Acad Sci US A. 2012 Jan 17; 109(3): 875-80.

[0078] Methods for preparing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (e.g., see Millstein et al., Nature, 305: 537-539 (1983)). Similar methods are disclosed in, for example, WO 93 / 08829, Traunecker et al., EMBO J., 10: 3655-3659 (1991), and also see Mouquet et al., "Enhanced HIV-1neutralization by antibody heteroligation" Proc Natl Acad Sci USA. 2012 Jan 17; 109 (3): 875-80.

[0079] Alternatively, the antibody variable region (antibody-antigen combination site) with the desired binding specificity is fused to the immunoglobulin constant domain sequence. The fusion is fused to the Ig heavy chain constant domain of at least the portion comprising hinge, CH2 and CH3 regions. According to some embodiments, there is a first heavy chain constant region (CH1) containing the required site for light chain bonding in at least one fusion (fusion). The DNA encoding the immunoglobulin heavy chain fusion and immunoglobulin light chain (if necessary) is inserted into a separate expression vector and co-transfected into a suitable host cell. When the unequal ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody, this provides greater flexibility in the embodiments to adjust the mutual ratio of the three polypeptide fragments. However, when the expression of the equal ratios of at least two polypeptide chains results in high yields, or when the ratio has no significant effect on the yield of the desired chain combination, it is possible to insert the coding sequence of two or all three polypeptide chains into a single expression vector.

[0080] The technology of producing bispecific antibodies from antibody fragments has also been described in the literature. For example, chemical bonds can be used to prepare bispecific antibodies. For example, Brennan et al., Science, 229: 81 (1985) described a method in which intact antibodies are proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the vicinal dithiols and prevent the formation of intermolecular disulfides. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted into a Fab'-thiol by reduction using mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibodies can be used as reagents for enzyme selective immobilization.

[0081] Other modifications of antibodies are contemplated herein. For example, the antibody can be linked to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyethylene oxide, or a copolymer of polyethylene glycol and polypropylene glycol. The antibody can also be embedded in a microcapsule, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively), in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in a coarse emulsion. These techniques are disclosed in, for example, Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0082] Typically, the antibodies of the invention are recombinantly produced using vectors and methods available in the art.Human antibodies can also be generated by in vitro activated B cells (see, for example, US Pat. Nos. 5,567,610 and 5,229,275). General methods of molecular genetics and genetic engineering that can be used in the present disclosure are described in the current editions of Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, D. Goeddel, ed., 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (MP Deutscher, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (RI Freshney. 1987. Liss, Inc. New York, NY), and Gene Transfer and Expression Protocols, pp. 109-128, EJ Murray, ed., The Humana Press Inc., Clifton, NJ). Reagents, cloning vectors, and kits for genetic manipulations are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0083] Human antibodies can also be produced in transgenic animals (e.g., mice) that can produce a complete pool of human antibodies in the absence of endogenous immunoglobulins. For example, homozygous deletions of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice have been described, resulting in complete suppression of endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies under antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all from GenPharm); U.S. Patent No. 5,545,807; and WO 97 / 17852. Such animals can be genetically engineered to produce human antibodies comprising the polypeptides of the invention.

[0084] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in or secreted from E. coli, making it easy to produce large quantities of these fragments. Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (see, e.g., Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab′)2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life comprising salvage receptor binding epitope residues are described in US Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to the skilled artisan.

[0085] Other techniques known in the art for selecting antibody fragments from libraries using enrichment techniques include, but are not limited to, phage display, ribosome display (Hanes and Pluckthun, 1997, Proc. Nat. Acad. Sci. 94: 4937-4942), bacterial display (Georgiou et al., 1997, Nature Biotechnology 15: 29-34) and / or yeast display (Kieke et al., 1997, Protein Engineering 10: 1303-1310) can be used as an alternative to the techniques discussed previously to select single-chain antibodies. Single-chain antibodies are selected from single-chain antibody libraries produced directly using filamentous phage technology. Phage display technology is known in the art (e.g., see the technology in Cambridge Antibody Technology (CAT)), as disclosed in U.S. Pat. Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255; 6,140,471; 6,225,447; 6,291650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081, and other U.S. family members, or applications relying on priority document GB9206318 filed May 24, 1992; see also Vaughn et al. 1996, Nature Biotechnology 14:309-314). Single-chain antibodies can also be designed and constructed using existing recombinant DNA techniques, such as DNA amplification methods (eg, PCR), or possibly by using the respective hybridoma cDNA as a template.

[0086] Variant antibodies are also included within the scope of the present invention. Therefore, variants of the sequences described in this application are also included within the scope of the present invention. Other variants of antibody sequences with improved affinity can be obtained using methods known in the art, and are included within the scope of the present invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve translation efficiency in expression systems for producing antibodies.

[0087] Such variant antibody sequences will share 70% or more (i.e., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher) sequence identity with the sequences disclosed in the present application. Such sequence identity is calculated relative to the full length of the reference sequence (i.e., the sequence described in the present application). As referred to herein, percent identity is determined using BLAST version 2.1.3 using the default parameters specified by NCBI (National Center for Biotechnology Information) [Blosum 62 matrix; gap open penalty (gap open penalty) = 11, gap extension penalty (gap extension penalty) = 1]. For example, the peptide sequences provided by the present disclosure comprise at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more continuous peptides of one or more sequences disclosed herein and all intervening lengths therebetween. As used herein, the term "intermediate length" is intended to describe any length between the recited values, for example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.

[0088] The present disclosure provides antibodies, alone or in combination with other antibodies, such as but not limited to VRCOl, anti-V3 loop, CD4b and CD4i antibodies, and PG9 / PG16-like antibodies, that have broad neutralizing activity in serum.

[0089] According to another embodiment, the present disclosure provides methods for preparing and administering HIV antibody compositions suitable for administration to a human or non-human primate patient having or at risk of HIV infection in an amount and on a schedule sufficient to induce a protective immune response against HIV or HIV viral reduction in humans.

[0090] According to another embodiment, the present disclosure provides a vaccine comprising at least one antibody of the present disclosure and a pharmaceutically acceptable carrier. According to one embodiment, the vaccine is a vaccine comprising at least one antibody described herein and a pharmaceutically acceptable carrier. The vaccine may comprise any combination of multiple antibodies having the characteristics described herein, and may further comprise antibodies known in the art that neutralize HIV.

[0091] It should be understood that the composition can be a single or combined form of the antibodies disclosed herein, which can be the same or different, to preventively or therapeutically treat the progression of HIV infection of various subtypes after vaccination. Such a combination can be selected based on the desired immunity. When the antibodies are administered to animals or humans, they can be combined with one or more pharmaceutically acceptable carriers, excipients or adjuvants known to those of ordinary skill in the art. The composition may further comprise broadly neutralizing antibodies known in the art, including but not limited to VRCO1, b12, anti-V3 loop, CD4b and CD4i antibodies, and PG9 / PG16-like antibodies.

[0092] In addition, with regard to determining the effective level of HIV treatment in patients, in particular, suitable animal models are available and have been widely used to evaluate the in vivo efficacy of various gene therapy regimens against HIV (Sarver et al. (1993b), see above). These models include mice, monkeys, and cats. Even though these animals are not naturally susceptible to HIV disease, chimeric mouse models (e.g., SCID, bg / nu / xid, NOD / SCID, SCID-hu, immunocompetent SCID-hu, myeloablated BALB / c) reconstituted with human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver / thymus, or other tissues can be infected with lentiviral vectors or HIV and used as models of HIV pathogenesis. Similarly, simian immunodeficiency virus (SIV) / monkey models can be used, as can feline immunodeficiency virus (FIV) / cat models. When used for the therapeutic treatment of AIDS, the pharmaceutical composition can contain other drugs, as well as the vector according to the present invention. These other drugs can be used in their traditional manner (i.e., as agents for treating HIV infection).

[0093] According to another embodiment, the present disclosure provides an antibody-based pharmaceutical composition comprising an effective amount of an isolated HIV antibody or affinity matured form, which provides a preventive or therapeutic treatment option to reduce HIV viral infection. The pharmaceutical composition may further comprise a second therapeutic agent. In some embodiments, the second therapeutic agent may be an anti-HIV-1 broadly neutralizing antibody. The anti-HIV-1 broadly neutralizing antibody can be one of the following: 10-259, 10-303, 10-410, 10-847, 10-996, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, 10-1074GM, GL, 10E8, 12A12, 12A21, 2F5, 2G12, 35022, 3BC176, 3BNC117, 3BNC55, 3BNC60, 3BNC62, 447-52D, 4E10, 5H / I1-BMV-D5, 8ANC195, b1 2. CAP256-VRC26.01, CAP256-VRC26.02, CAP256-VRC26.03, CAP256-VRC26.04, CAP256-VRC26.05, CAP256-VRC26.06, CAP256-V RC26.07, CAP256-VRC26.08, CAP256-VRC26.09, CAP256-VRC26.10, CAP256-VRC26.11, CAP256-VRC26.12, CH01, CH02, CH03, CH04 , CH103, HGN194, HJ16, HK20, M66.6, NIH45-46, PCDN-33A, PCDN-33B, PCDN-38A, PG9, PG16, PGDM1400, PGDM1401, PGDM1402, PGDM 1403, PGDM1404, PGDM1405, PGDM1406, PGDM1407, PGDM1408, PGDM1409, PGDM1410, PGDM1411, PGDM1412, PGT121, PGT122, PGT123 ,PGT125,PGT126,PGT127,PGT128,PGT130,PGT131,PGT135,PGT136,PGT137,PGT141,PGT142,PGT143,PGT145,PGT151,PGT152, VRC-CH30, VRC-CH31, VRC-CH32, VRC-CH33, VRC-CH34, VRC-PG04, VRC-CH04b, VRC-PG20, VRC01, VRC02, VRC03, VRC07, VRC23 and Z13.In some embodiments, the broadly neutralizing anti-HIV-1 antibody is 3BNC117. 3BNC117 is a next-generation bNAb that targets the CD4 binding site on the HIV envelope gp160. It is a recombinant human IgG1 kappa monoclonal antibody cloned from a viremic HIV-infected controller. A long-acting version of 3BNC117 is called 3BNC117-LS. 3BNC117 is described in U.S. Patent No. 9,783,594.

[0094] The antibody-based pharmaceutical compositions of the present disclosure can be formulated by a number of strategies known in the art (e.g., see McGoff and Scher, 2000, Solution Formulation of Proteins / Peptides: In McNally, EJ, ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, PA: Talyor and Francis; pp. 145-177; Akers et al., 2002, Pharm. Biotechnol. 14: 47-127). Pharmaceutically acceptable compositions suitable for administration to a patient will contain an effective amount of the antibody in a formulation that both retains biological activity and promotes maximum stability during storage within an acceptable temperature range. Depending on the desired formulation, the pharmaceutical composition may also include a pharmaceutically acceptable diluent, a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient, or any such vehicle commonly used in the preparation of pharmaceutical compositions for animal or human administration. A diluent that does not affect the biological activity of the composition is selected. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hanks' solution. The amount of excipient that can be used in the pharmaceutical composition or formulation of the present invention is such that the antibody is evenly distributed throughout the composition so that it can be evenly dispersed when delivered to a subject in need thereof. It can be used to dilute the antibody to a concentration that provides the desired beneficial relief or therapeutic result while minimizing any adverse side effects that may arise from excessive concentrations. It can also have a preservative effect. Therefore, for antibodies with high physiological activity, more excipients will be used. On the other hand, for any active ingredient that exhibits lower physiological activity, a smaller amount of excipient will be used.

[0095] The above-described antibodies and antibody compositions or vaccine compositions comprising at least one antibody described herein or a combination thereof can be administered for the prophylactic and therapeutic treatment of HIV viral infection.

[0096] The present disclosure further relates to isolated polypeptides, which include the new amino acid sequences of the light chain district and heavy chain variable region listed in Table 2-3. In other related embodiments, the present disclosure provides polypeptide variants with the amino acid sequence of the light chain district and heavy chain variable region of HIV antibodies, as determined using the method described herein (i.e., BLAST analysis using standard parameters), which have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity compared to the peptide sequence listed in Table 2-3. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of the encoded protein by considering amino acid similarity, etc. In other related embodiments, the present disclosure provides polypeptide variants having amino acid sequences of light chain regions and heavy chain variable regions of HIV antibodies having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity compared to the polypeptide sequences listed in Tables 2-3, and having amino acid sequences of CDR regions that are identical or substantially identical to those listed in Table 4 or identical or substantially identical to the amino acid sequences of the CDR regions of the unmodified 10-1074-LS antibody (or MS-193). In other related embodiments, the present disclosure provides polypeptide variants having amino acid sequences of light chain regions and heavy chain variable regions of HIV antibodies having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity compared to the polypeptide sequences listed in Tables 2-3, and having amino acid sequences of CDR regions that are identical or substantially identical to those listed in Table 4 or identical or substantially identical to the amino acid sequences of the CDR regions of the unmodified 10-1074-LS antibody (or MS-193), such that the polypeptide variants retain 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher binding affinity to HIV virus. The term "substantially identical" refers to a sequence that is greater than about 85% identical to another sequence.

[0097] The term "polypeptide" is used in its conventional sense, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific length of the product. Peptides, oligopeptides, and proteins are all included within the definition of polypeptide, and these terms are used interchangeably herein unless otherwise expressly stated. This term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other naturally occurring and non-naturally occurring modifications known in the art. The polypeptide can be a complete protein or a subsequence thereof. In the context of the present disclosure, a specific polypeptide of interest is an amino acid subsequence comprising CDRs, VH, and VL and capable of binding to an antigen or a cell infected by HIV.

[0098] As used herein, the term "variant" of a polypeptide is a polypeptide that generally differs from the polypeptides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more of the above-disclosed polypeptide sequences and evaluating one or more biological activities of the polypeptides described herein and / or using any of a number of techniques known in the art.

[0099] For example, certain amino acids in a protein structure can be replaced by other amino acids without significantly losing their ability to bind to other polypeptides (e.g., antigens) or cells. Since it is the binding capacity and properties of the protein that determine the biological function activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, accordingly, in the DNA coding sequence of its basis to obtain proteins with similar properties. Therefore, it is expected that various changes can be made to the peptide sequence of the disclosed composition or the corresponding DNA sequence encoding the peptide without significantly losing its biological use or activity.

[0100] Variant antibody sequences include those into which conservative substitutions have been introduced by modifying the polynucleotides encoding the polypeptides of the present invention. Amino acids can be classified based on their physical properties and contribution to secondary and tertiary protein structure. In the art, a "conservative substitution" is considered to be the replacement of one amino acid with another amino acid having similar properties. Exemplary conservative substitutions are shown below:

[0101]

[0102] Alternatively, conserved amino acids may be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77] as follows:

[0103]

[0104]

[0105] Alternatively, exemplary conservative substitutions are shown below:

[0106]

[0107]

[0108] A conservative substitution of an existing substitution refers to a conservative substitution of the substituted residue. For example, a conservative substitution of LmdV:Y2P refers to a conservative substitution of proline (P) at position LmdV:Y2 (i.e., glycine (G)). In another example, a conservative substitution of HV:V79T refers to a conservative substitution of threonine (T) at position HV:V79 (i.e., serine (S), cysteine ​​(C)).

[0109] "Homology" or "sequence identity" refers to the percentage of residues in a polynucleotide or polypeptide sequence variant that are identical to the non-variant sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of homology. In specific embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.

[0110] Such variant polypeptide sequences will have 70% or more (i.e. 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences described herein. In other embodiments, the invention provides polypeptide fragments of various lengths of continuous extensions comprising the amino acid sequences disclosed herein. For example, the disclosure provides peptide sequences comprising at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more continuous peptides of one or more sequences disclosed herein and all intervening lengths.

[0111] The present disclosure also includes nucleic acid sequences encoding part or all of the light and heavy chains of the inventive antibodies and fragments thereof. Due to the redundancy of the genetic code, there will be variants of these sequences encoding the same amino acid sequence.

[0112] The present disclosure also includes the light chain of the HIV antibody listed in coding table 2-3 and the nucleic acid sequence of the separation of the polypeptide of heavy chain.In other related embodiments, described invention provides the heavy chain of the HIV antibody listed in coding table 5-6 and the polynucleotide variants of the peptide sequence of light chain.As determined using method as herein described (that is, using the BLAST analysis of standard parameters), these polynucleotide variants have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or higher sequence identity compared with the polynucleotide sequence of the present disclosure.Those skilled in the art will recognize that these values ​​can be suitably adjusted to determine the corresponding identity of the protein encoded by two nucleotide sequences by considering codon degeneracy, amino acid similarity, reading frame positioning etc.

[0113] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to single-stranded or double-stranded RNA, DNA, or mixed polymers. Polynucleotides can include genomic sequences, extragenomic and plasmid sequences, and smaller engineered gene segments that express or can be adapted to express a polypeptide.

[0114] An "isolated nucleic acid" is a nucleic acid that is substantially separated from other genomic DNA sequences and proteins or complexes that naturally accompany the native sequence, such as ribosomes and polymerases. The term encompasses nucleic acid sequences that have been removed from their naturally occurring environment and includes recombinant or cloned DNA isolates as well as chemically synthesized analogs or analogs biologically synthesized by heterologous systems. Substantially pure nucleic acid includes isolated forms of the nucleic acid. Thus, this refers to the initially isolated nucleic acid and does not exclude genes or sequences that have been subsequently added to the isolated nucleic acid by humans.

[0115] As used herein, the term polynucleotide "variant" is a polynucleotide that generally differs from the polynucleotides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more polynucleotide sequences disclosed herein and evaluating one or more biological activities of the encoded polypeptides described herein and / or using any of a number of techniques known in the art.

[0116] The structure of the polynucleotides of the invention can be modified and still obtain functional molecules encoding variants or derivative polypeptides having desired characteristics. When it is necessary to change the amino acid sequence of a polypeptide to create an equivalent or even improved variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more codons of the encoding DNA sequence.

[0117] Typically, polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions such that the immunogenic binding properties of the polypeptide encoded by the variant polynucleotide are not substantially diminished compared to the polypeptide encoded by the polynucleotide sequence specifically set forth herein.

[0118] In other embodiments, the invention provides polynucleotide fragments of various lengths comprising sequences identical or complementary to one or more sequences disclosed herein. For example, the disclosure provides polynucleotides comprising at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more consecutive nucleotides of one or more sequences disclosed herein, as well as all intervening lengths therebetween, and encompassing any length between the recited values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; and including all integers between 200-500; 500-1,000.

[0119] In another embodiment of the present invention, a polynucleotide composition is provided that can hybridize to a polynucleotide sequence provided herein or a fragment thereof or its complementary sequence under medium to high stringency conditions. Hybridization techniques are well known in the art of molecular biology. For illustrative purposes, suitable medium stringency conditions for testing the hybridization of the polynucleotides disclosed herein to other polynucleotides include pre-washing in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50-60°C, 5x SSC; and then washing twice with 2x, 0.5x and 0.2x SSC containing 0.1% SDS at 65°C for 20 minutes each. It will be understood by those skilled in the art that the stringency of hybridization can be easily manipulated by, for example, changing the salt content of the hybridization solution and / or the temperature at which hybridization is performed. For example, in another embodiment, suitable high stringency hybridization conditions include those described above, except that the hybridization temperature is increased to, for example, 60-65°C or 65-70°C.

[0120] In some embodiments, the polypeptides encoded by the polynucleotide variants or fragments have the same binding specificity as the polypeptides encoded by the native polynucleotides (i.e., specifically bind or preferentially bind to the same epitope or HIV strain). In some embodiments, the level of binding activity of the polypeptides encoded by the polynucleotides, polynucleotide variants, fragments, and hybridizing sequences is at least about 50%, at least about 70%, and at least about 90% of the binding activity level of the polypeptide sequences specifically set forth herein.

[0121] The polynucleotides or fragments thereof of the invention, regardless of the length of the coding sequence itself, can be combined with other DNA sequences such as promoters, polyadenylation signals, other restriction enzyme sites, multiple cloning sites, other coding segments, etc., so that their total length can be greatly varied. Nucleic acid fragments of almost any length are employed. For example, exemplary polynucleotide segments having a total length of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, etc. (including all intervening lengths) are included in many embodiments of the invention.

[0122] The scope of the present invention further includes vectors, such as expression vectors, comprising the nucleic acid sequences of the present invention. Cells transformed with such vectors are also within the scope of the present invention.

[0123] The disclosure also provides vectors and host cells comprising nucleic acids of the present invention, and recombinant techniques for producing polypeptides of the present invention. Vectors of the present invention include those that can replicate in any type of cell or organism, including, for example, plasmids, phages, cosmids, and minichromosomes. In some embodiments, the vector comprising the polynucleotide of the invention is a vector suitable for propagation or replication of the polynucleotide, or a vector suitable for expressing the polypeptide of the invention. Such vectors are known in the art and are commercially available.

[0124] "Vectors" include shuttle vectors and expression vectors. Typically, plasmid constructs will also include an origin of replication (e.g., the ColE1 origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance) for plasmid replication and selection in bacteria, respectively. "Expression vectors" refer to vectors that contain the necessary control sequences or regulatory elements for expression of antibodies, including antibody fragments of the present invention, in bacteria or eukaryotic cells.

[0125] As used herein, the term "cell" can be any cell, including but not limited to cells of eukaryotic, multicellular species (e.g., as opposed to unicellular yeast cells), such as, but not limited to, mammalian cells or human cells. A cell can exist as a single entity, or can be part of a larger cell collection. Such a "larger cell collection" can include, for example, a cell culture (mixed or pure), a tissue (e.g., endothelial, epithelial, mucosal or other tissue), an organ (e.g., lung, liver, muscle and other organs), an organ system (e.g., circulatory system, respiratory system, gastrointestinal system, urinary system, nervous system, epidermal system or other organ system), or an organism (e.g., avian, mammalian, etc.).

[0126] Polynucleotide of the present invention can be synthesized as a whole or with part synthesis and then combined, and use conventional molecular and cell biology techniques to be inserted into a vector, including, for example, using suitable restriction sites and restriction enzymes that the polynucleotide is subcloned into a linearized vector. Use oligonucleotide primers complementary to each chain of the polynucleotide to increase the polynucleotide of the invention by polymerase chain reaction. These primers also include restriction enzyme sites to promote subcloning into a vector. Replicable vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, and one or more markers or selectable genes.

[0127] In order to express the polypeptide of the present invention, the nucleotide sequence encoding the polypeptide or a functional equivalent can be inserted into a suitable expression vector, i.e., a vector containing the elements required for the inserted coding sequence for transcription and translation. Methods well known to those skilled in the art can be used to construct an expression vector containing a sequence encoding the polypeptide of interest and suitable transcription and translation control elements. These methods include in vitro recombinant DNA technology, synthetic techniques, and in vivo genetic recombination. Such technology is described in, for example, Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY and Ausubel, FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York.NY.

[0128] The present disclosure also provides kits that can be used for diagnosis and prognosis determination using the antibodies, polypeptides and nucleic acids of the present invention. The kits of the present invention include suitable containers containing HIV antibodies, polypeptides or nucleic acids of the present invention in labeled or unlabeled form. In addition, when the antibodies, polypeptides or nucleic acids are provided in a labeled form suitable for indirect binding determination, the kit further includes reagents for performing suitable indirect determinations. For example, depending on the nature of the label, the kit may include one or more suitable containers containing enzyme substrates or derivatizing agents. Control samples and / or instructions may also be included. The present disclosure also provides kits for detecting the presence of HIV antibodies of the present disclosure or nucleotide sequences of HIV antibodies in biological samples by PCR or mass spectrometry.

[0129] In some embodiments, the kit comprises a pharmaceutically effective amount of at least one pharmaceutically acceptable dosage unit of an isolated anti-HIV antibody or antigen-binding portion thereof as described herein. The kit may further comprise a pharmaceutically effective amount of a pharmaceutically acceptable dosage unit of an anti-HIV agent. The two pharmaceutically acceptable dosage units may optionally be in the form of a single pharmaceutically acceptable dosage unit. Exemplary anti-HIV agents may be selected from the group consisting of non-nucleoside reverse transcriptase inhibitors, protease inhibitors, entry or fusion inhibitors, and integrase inhibitors. In some embodiments, the anti-HIV agent is an anti-HIV broadly neutralizing antibody, such as 3BNC117.

[0130] As used herein, "label" refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody to produce a "labeled" antibody. Labels can also be conjugated to the polypeptides and / or nucleic acid sequences disclosed herein. The label can itself be detectable (e.g., a radioisotope label or a fluorescent label), or for enzymatic labels, can catalyze a chemical change in a detectable substrate compound or composition. The antibodies and polypeptides of the invention can also be modified to include an epitope tag or label, for example for purification or diagnostic applications. Suitable detection means include the use of labels such as, but not limited to, radionucleotides, enzymes, coenzymes, fluorescent agents, chemiluminescent agents, chromophores, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like.

[0131] According to another embodiment, the present disclosure provides a diagnostic method. The diagnostic method generally involves contacting a biological sample obtained from a patient (e.g., blood, serum, saliva, urine, sputum, cell swab sample, or tissue biopsy) with HIV antibodies and determining whether the antibody preferentially binds compared to a control sample or a predetermined cutoff value, thereby indicating the presence of HIV virus.

[0132] According to another embodiment, the present disclosure provides a method for detecting the presence of HIV antibodies of the present disclosure in a biological sample from a patient. The detection method generally involves obtaining a biological sample (e.g., blood, serum, saliva, urine, sputum, cell swab sample, or tissue biopsy) from the patient, and isolating HIV antibodies or fragments thereof or nucleic acids encoding HIV antibodies, and determining the presence of HIV antibodies in the biological sample. The present disclosure also provides a method for detecting the nucleotide sequence of HIV antibodies in a cell. Primers disclosed herein can also be used to detect the nucleotide sequence of HIV antibodies. The presence of HIV antibodies in a biological sample from a patient can be determined by utilizing known recombinant techniques and / or using a mass spectrometer.

[0133] In another embodiment, the present disclosure provides a method for detecting HIV antibodies in a biological sample, wherein the HIV antibodies comprise a heavy chain containing a highly conserved consensus sequence and a light chain containing a highly conserved consensus sequence, the method comprising obtaining a biological sample containing immunoglobulins from a mammalian subject, isolating HIV antibodies from the sample, and identifying the highly conserved consensus sequences of the heavy and light chains. The biological sample can be blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy. The amino acid sequence can be determined by methods known in the art, including, for example, PCR and mass spectrometry.

[0134] The term "assessing" includes any form of measurement and includes determining whether an element is present. The terms "determine," "measure," "evaluate," "assess," and "determine" are used interchangeably and include both quantitative and qualitative determinations. An assessment can be relative or absolute. "Assessing the presence of" includes determining the amount of something present and / or determining whether it is present. As used herein, the terms "determine," "measure," "assess," and "determine" are used interchangeably and include both quantitative and qualitative determinations.

[0135] Methods to reduce viral replication

[0136] Further provided are methods for reducing HIV viral titer, viral replication, viral proliferation, or an increase in the amount of HIV viral protein in a subject. According to another aspect, the method comprises administering to a subject an HIV antibody effective to reduce HIV titer, viral replication, or an increase in the amount of HIV protein of one or more HIV strains or isolates in the subject.

[0137] According to another embodiment, the present disclosure provides a method of reducing viral replication or spread of HIV infection to other host cells or tissues, comprising contacting a mammalian cell with an antibody or portion thereof that binds to an antigenic epitope on gp120.

[0138] Treatment

[0139] According to another embodiment, the present disclosure provides a method for treating a mammal infected with a virus, such as HIV, comprising administering to the mammal a pharmaceutical composition comprising an HIV antibody disclosed herein. According to one embodiment, the method for treating a mammal infected with HIV comprises administering to the mammal a pharmaceutical composition comprising an antibody or fragment thereof disclosed herein. The composition of the present disclosure may comprise more than one antibody having the disclosed characteristics (e.g., a plurality or a plurality of antibodies).It may also include other HIV neutralizing antibodies known in the art, such as, but not limited to, 10-259, 10-303, 10-410, 10-847, 10-996, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, 10-1074GM, GL, 10E8, 12A12, 12A21, 2F5, 2G12, 35022, 3BC176, 3BNC117, 3BNC55, 3BNC60, 3BNC62, 447-52D, 4E10, 5H / I1-BMV-D5, 8ANC 195, b12, CAP256-VRC26.01, CAP256-VRC26.02, CAP256-VRC26.03, CAP256-VRC26.04, CAP256-VRC26.05, CAP256-VRC26.06, CAP 256-VRC26.07, CAP256-VRC26.08, CAP256-VRC26.09, CAP256-VRC26.10, CAP256-VRC26.11, CAP256-VRC26.12, CH01, CH02, CH03, CH04, CH103, HGN194, HJ16, HK20, M66.6, NIH45-46, PCDN-33A, PCDN-33B, PCDN-38A, PG9, PG16, PGDM1400, PGDM1401, PGDM1402, P GDM1403, PGDM1404, PGDM1405, PGDM1406, PGDM1407, PGDM1408, PGDM1409, PGDM1410, PGDM1411, PGDM1412, PGT121, PGT122, PGT1 23. PGT125, PGT126, PGT127, PGT128, PGT130, PGT131, PGT135, PGT136, PGT137, PGT141, PGT142, PGT143, PGT145, PGT151, PGT152 , VRC-CH30, VRC-CH31, VRC-CH32, VRC-CH33, VRC-CH34, VRC-PG04, VRC-CH04b, VRC-PG20, VRC01, VRC02, VRC03, VRC07, VRC23 and Z13.

[0140] The method may further include administering a second therapeutic agent, such as a therapeutically effective amount of a second therapeutic agent. The second therapeutic agent may be administered before, simultaneously with, or after the administration of the anti-HIV antibody or antigen-binding portion thereof. In some embodiments, the second therapeutic agent is an anti-HIV-1 broadly neutralizing antibody. Examples of anti-HIV-1 broadly neutralizing antibodies are provided above. In some embodiments, the anti-HIV-1 broadly neutralizing antibody is 3BNC117.

[0141] Passive immunization has been shown to be an effective and safe strategy for preventing and treating viral diseases. (See, e.g., Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashi et al., Nat. Med. 5:211-16 (1999). Passive immunization using human monoclonal antibodies provides an immediate therapeutic strategy for the emergency prevention and treatment of HIV.

[0142] Subjects at risk of contracting an HIV-related disease or condition include patients who have come into contact with an infected person or have been exposed to HIV in some other way. Prophylactic agents can be administered before the onset of symptoms characteristic of an HIV-related disease or condition, thereby preventing or delaying the progression of the disease or condition.

[0143] In order to treat in vivo to humans and non-human patients, a pharmaceutical preparation comprising HIV antibodies of the present disclosure is administered or provided to the patient. When used for in vivo therapy, antibodies of the present disclosure are administered to the patient in a therapeutically effective amount (i.e., the amount that eliminates or alleviates the patient's viral load). According to known methods, antibodies are administered to human patients, such as intravenously, such as bolus injection or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, local or inhalation routes. Antibodies can be administered parenterally (if possible) or intravenously at the target cell site. In some embodiments, antibodies are administered by intravenous or subcutaneous administration. The therapeutic composition of the present disclosure can be administered to a patient or subject systemically, parenterally or topically. The above-mentioned parameters for successful treatment and improvement of the disease for assessment can be easily measured by the routine procedures familiar to the physician.

[0144] For parenteral administration, the antibody can be formulated into a unit dose injectable form (solution, suspension, emulsion) with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles include, but are not limited to, fixed oils and ethyl oleate. Liposomes can be used as carriers. The vehicle may contain a small amount of additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. The antibody can be formulated in such a vehicle at a concentration of about 1 mg / ml to 150 mg / ml.

[0145] Dosage and dosage regimen depend on the multiple factors that the doctor is easy to determine, such as the character of infection, such as its therapeutic index, patient and patient's medical history.Usually, the antibody of therapeutically effective amount is administered to the patient.In some embodiments, the antibody amount range of administration is about 0.1mg / kg to about 50mg / kg of patient body weight.Depending on the type and severity of infection, the antibody is administered to the patient with an initial candidate dose of about 0.1mg / kg to about 50mg / kg body weight (for example, about 0.1-15mg / kg / dosage), either by, for example, one or more separate administrations, or by continuous infusion.The progress of this therapy can be easily monitored by conventional methods and assays, and is based on standards known to doctors or other persons skilled in the art.The above-mentioned parameters for successful treatment and improvement for assessing disease can be easily measured by the conventional procedures familiar to doctors.

[0146] Other treatment regimens can be combined with the administration of the HIV antibodies disclosed herein. Co-administration includes co-administration using separate formulations or single pharmaceutical formulations, as well as continuous administration in any order, wherein preferably both (or all) active agents exert their biological activities simultaneously over a certain period of time. Such combination therapy can produce a synergistic therapeutic effect. The parameters described above for evaluating successful treatment and improvement of the disease can be easily measured by routine procedures familiar to physicians.

[0147] The terms "treat" or "alleviate" are used interchangeably and refer to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the target pathological condition or disorder. Those in need of treatment include those already suffering from the disorder as well as those susceptible to developing the disorder or those for which the disorder is to be prevented. An infection in a subject or mammal is successfully "treated" if, after receiving a therapeutic amount of an antibody according to the methods of the present disclosure, the patient shows an observable and / or measurable reduction or absence of one or more of the following: a decrease in the number of infected cells or an absence of infected cells; a decrease in the percentage of the total number of infected cells; and / or a alleviation to some extent of one or more symptoms associated with the specific infection; a decrease in morbidity and mortality, and an improvement in quality of life issues. The above-mentioned parameters for assessing successful treatment and improvement of the disease can be readily measured by routine procedures familiar to physicians.

[0148] The terms "effective amount," "effective dose," or "effective dosage" are defined as an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of impairment or disability resulting from the affliction of the disease. A "prophylactically effective amount" or "prophylactically effective dose" of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering a recurrence of the disease, inhibits the progression or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit disease progression or recurrence can be assessed using a variety of methods known to those skilled in the art, for example, in human subjects during clinical trials, in animal model systems that are predictive of efficacy in humans, or by measuring agent activity in in vitro assays.

[0149] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0150] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are typically aqueous pH buffered solutions. Examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants, including, but not limited to, ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as, but not limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as, but not limited to, polyvinylpyrrolidone; amino acids, such as, but not limited to, glycine, glutamine, asparagine, arginine, proline, or lysine; monosaccharides, disaccharides, and other carbohydrates, including, but not limited to, glucose, mannose, or dextrin; chelating agents, such as, but not limited to, EDTA; sugar alcohols, such as, but not limited to, mannitol, sorbitol, sucrose, or trehalose; salt-forming counterions, such as, but not limited to, sodium; and / or nonionic surfactants, such as, but not limited to, TWEEN.; polyethylene glycol (PEG); poloxamer, i.e., Pluronic F-68. F-68), and polysorbates, namely polysorbate 20 or polysorbate 80.

[0151] definition

[0152] In order to help understand the detailed description of the compositions and methods described in the present disclosure, some clear definitions are provided to promote the clear disclosure of various aspects of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0153] When referring to nucleic acid molecules, the term "recombinant" refers to nucleic acid molecules composed of nucleic acid segments joined together by means of molecular biological techniques. When referring to proteins or polypeptides, the term "recombinant" refers to protein molecules expressed using recombinant nucleic acid molecules.

[0154] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0155] As used herein, the term "in vitro" refers to events that occur in an artificial environment (eg, in a test tube or reaction vessel, in cell culture, etc.) rather than in a multicellular organism.

[0156] As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a non-human animal.

[0157] The terms "prevent," "prophylactic treatment," and the like refer to reducing the likelihood of a disease or condition developing in a subject who does not already have it but is at risk of or susceptible to developing the disease or condition.

[0158] As used herein, "administering" refers to the physical introduction of a composition comprising a therapeutic agent into a subject using various methods and delivery systems known to those skilled in the art. Routes of administration as described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, typically by injection, and includes but is not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, compositions as described herein can be administered by non-parenteral routes, such as topical, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, repeatedly and / or in one or more persistent phases.

[0159] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule (e.g., a nucleic acid, an antibody, a protein, or a portion thereof, such as a peptide), or an extract made from biological material, such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues. The activity of such an agent may make it suitable as a "therapeutic agent," which is one or more substances with biological, physiological, or pharmacological activity that act locally or systemically in a subject.

[0160] The terms "therapeutic agent," "therapeutic capable agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect when administered to a subject. Beneficial effects include achieving a diagnostic determination; ameliorating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and overall counteracting a disease, symptom, disorder, or pathological condition.

[0161] Unless the context clearly indicates otherwise, "combination" therapy as used herein is intended to encompass the administration of two or more therapeutic agents in a coordinated manner, and includes, but is not limited to, simultaneous administration. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation, or simultaneous administration of separate therapeutic compositions) and continuous or sequential administration, provided that the administration of one therapeutic agent is in some way dependent on the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a specified period of time. See, e.g., Kohrt et al. (2011) Blood 117:2423.

[0162] When range values ​​are provided, it is understood that unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of the range, to one-tenth of the unit of the lower limit, and any other specified or intervening values ​​in the specified range are encompassed within the present disclosure. The upper and lower limits of these smaller ranges that may independently be included in the smaller ranges are also encompassed within the present disclosure, not including any specifically excluded limit in the specified range. When a specified range includes one or both limits, ranges excluding either of those included limits are also encompassed within the present disclosure.

[0163] It must be noted herein that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0164] Unless stated otherwise, the terms "including," "comprising," "containing," or "having" and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter.

[0165] Repeated use of the phrases "in one embodiment," "in various embodiments," "in some embodiments," etc. does not necessarily refer to the same embodiment, but they may, unless the context dictates otherwise.

[0166] The term "and / or" or " / " means any one of the items, any combination of these items, or all of the items related to this term.

[0167] The word "substantially" does not exclude "completely", for example, a composition "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0168] As used herein, the term "about" or "approximately", such as applied to one or more values ​​of interest, refers to a value similar to a specified reference value. In some embodiments, the term "about" or "approximately" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of either direction (greater than or less than) of a specified reference value, unless otherwise noted or apparent from the context (unless this number exceeds 100% of the possible value). Unless otherwise indicated herein, the term "about" is intended to include values ​​close to the range, such as weight percentages, which are equivalent with respect to the functionality of a single component, composition or embodiment.

[0169] As used herein, the term "each" when applied to a collection of items is intended to identify an individual item in the collection, but does not necessarily refer to every item in the collection, unless otherwise clearly indicated by the explicit disclosure or context.

[0170] Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element is essential to the practice of the invention.

[0171] Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein are performed in any suitable order. With respect to any method provided, the steps of the method can occur simultaneously or sequentially. When the steps of the method occur sequentially, these steps can occur in any order unless otherwise indicated.

[0172] Where the method comprises a combination of steps, each and every combination or subcombination of steps is encompassed within the scope of the present disclosure unless otherwise indicated herein.

[0173] Each publication, patent application, patent, or other reference cited herein is incorporated by reference in its entirety to the extent not inconsistent with the present disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. In addition, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

[0174] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in view thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims.

[0175] Example 1

[0176] 1 Identification and characterization of variants of 0-1074 broadly neutralizing antibodies - Round 1

[0177] Transient expression in HEK293 cells was used to generate the first round of variants, as shown in Table 9, including MS-203, MS-204, MS-205, MS-206, MS-207, MS-208, MS-209, MS-210, MS-211, MS-212, MS-213, MS-214, MS-215, MS-216, MS-217, MS-218, MS-219, MS-220, and MS-224, and purified by protein A chromatography. Characterization methods for analyzing variants are listed in Table 8, including size exclusion chromatography (SEC), differential scanning fluorimetry (DSF), low pH stability, and relative solubility assay (RSA). The antibody buffer was exchanged into phosphate buffered saline and used for analysis. Assays used to analyze the first round of variants included: SEC, for quantification of monomeric and high molecular weight species after purification; DSF, for characterization of CH2 and Fab domain stability during thermal ramping, and retention of neutralization capacity.

[0178] The range of the monomer content of variants is as low as 60.8%, up to 96.3%. The monomer content of unmodified 10-1074-LS (or MS-194) is 91.5%, and the remaining substances of all variants are high molecular weight substances (HMW). Consider using variants with HMW less than 10% for the second round of combination variants. In addition to SEC analysis, differential scanning fluorimetry is also used to define molecules with increased thermodynamic stability. For 10-1074-LS (or MS-194) parent molecules, only a single Tm is measured, indicating that CH2 and Fab domains unfold at the same temperature. For some variants, similar results are observed. However, a minority also shows the presence of Tm1 and a second melting transition (melting transition) called Tm2, ​​because modifications that contribute to stabilizing the Fab domains have been carried out in the Fv domain of the antibody, thereby resulting in an increase in thermal transition. It is not considered that the antibody that does not show consistent Tm2 in two repeated experiments analyzed by DSF is used for the second round of combination.

[0179] Neutralization activity was also measured to ensure that the activity of bnAb variants was retained. Table 10 shows the results of neutralization for six kinds of HIV pseudoviruses (such as Du156.12, WITO4160.33, CNE17, CNE30, CAAN5342.A2, Du172.17), which represent a wider range of viral groups with activity for 10-1074. It is believed that the IC50 or IC80 values ​​of specific pseudoviruses increased by more than 3 times are inactive, and are no longer considered further. Data prove that only one variant MS-208 loses neutralization activity, and is not selected for further development.

[0180] The final amino acid set for further development was based on a combination of: purified amount, high molecular weight percentage, improvement in thermodynamic stability (by DSF analysis), and retention of neutralizing activity. An example of the reasons for selecting residues for combinatorial analysis is described in Table 11. The five amino acid residues selected for further development were MS-203 (LmdV: Y2P), MS-216 (HV: V79T), MS-217 (HV: R82V), MS-218 (HV: L89F), and MS-219 (HV: T108R).

[0181] Example 2

[0182] Identification and characterization of variants of 10-1074 broadly neutralizing antibodies—round 2

[0183] The second round of combination variants was designed based on the first round variant described in the preceding section. The combination variants tested in the second round of optimization are shown in Table 12, and are composed of ten double combinations (double combination), ten triple combinations (triple combination), five quadruplicates and one quintuple combination (composed of whole five amino acid modifications). These variants include MS-200, MS-201, MS-202, MS-225, MS-226, MS-227, MS-228, MS-229, MS-230, MS-231, MS-232, MS-233, MS-234, MS-235, MS-236, MS-237, MS-238, MS-239, MS-240, MS-241, MS-242, MS-243, MS-244 and MS-245. Combination variants are produced using the transient expression in HEK293 cells, and purified by protein A chromatography. Prior to analysis, the antibody buffer was exchanged into phosphate-buffered saline. Assays used to analyze the second round of variants included: SEC for quantification of monomeric and high molecular weight species after purification; differential scanning fluorimetry for characterization of CH2 and Fab domain stability during thermal ramping; chemical unfolding, low pH stability, solubility, and retention of neutralization capacity.

[0184] The results of the preliminary screening consisting of SEC analysis of dimer and oligomer content and DSF for increased thermodynamic stability are shown in Table 13. For example, MS-200 has a lower HMW than the control variant MS-194. MS-200 also has Tm1=70.15°C and Tm2=74.62°C, indicating that it has improved thermostability. Separating the HMW species into dimer and oligomer species (wherein the HMW species elutes earlier than the dimer) provides a more accurate data view. The data show that the dimer content is relatively unchanged relative to 10-1074-LS (or MS-194), while the oligomer content of some variants has increased by up to 2 times, and the oligomer content of other variants has decreased by up to about 7 times. These variants are also characterized by DSF to identify those variants with increased thermodynamic stability (proven by the presence of different Tm2 unfolding temperatures).

[0185] In order to distinguish variants better by DSF, the alternative data analysis of the variation between Tm1 and Tm2 and the area under the thermal unfolding curve is designed. As shown in the data called DSF shoulder score (DSF Shoulder Score) in Table 14, these variants can have similar Tm2 values, but have different shoulder score values, and the higher the value, the higher the stability is. For example, the DSF shoulder score values ​​of MS-200, MS-201 and MS-202 are respectively 16.12, 29.39 and 22.49, which are significantly greater than the shoulder score value 7.65 of control antibody variant MS-194, showing that variant MS-200, MS-201 and MS-203 are more stable than control antibody variant MS-194. Thermodynamic stability has also been assessed by chemical unfolding, which has assessed the inherent resistance of native state to unfolding (measured by the midpoint of the denaturation curve). The higher the value, the higher the stability. Together with the DSF shoulder score, the intrinsic thermodynamic stability of antibody has been more accurately distinguished. In addition to inherent stability, the variants were also analyzed for resistance to aggregation, neutralization, and solubility under low pH incubation. While the parent 10-1074-LS (or MS-194) aggregated with up to 40% HMW formation, some variants showed only 2-3% HMW formation. The solubility of some variants was also increased, with solubility increased by up to 42% compared to the parent molecule.

[0186] The neutralizing ability of the subgroups of the combined variants was also checked to ensure that neutralization loss did not occur. As shown in Table 15, a reduced set of variants was tested against a group of 12 representative pseudoviruses (including SC422661.8, WITO4160.33, CAAN5342.A2, DU156.12, DU172.17, CNE17, CNE30, CNE53, 235-47, X1193_c1, X1254_c3 and 3301.v1.c24). Variants with a Tm of 2 were selected for testing. Among the tested variants, they all retained neutralizing activity against the pseudovirus group being examined.

[0187] Since all of the reduced set of antibodies defined in Table 15 retained neutralizing activity, the final set of variants that underwent in-depth biophysical analysis was defined based on biophysical properties. The specific reasons for excluding bnAbs from the set that underwent in-depth analysis are described in Table 16, and the final set is shown below.

[0188]

[0189]

[0190] In-depth analysis of the final variant set

[0191] The final optimized variants were based on the final variant set defined above. The analyses performed were downstream purification (Figures 1-2), accelerated stability (Figures 3-4). For downstream purification analysis and accelerated stability, molecules were produced using transient expression in the CHO-S cell line.

[0192] Results from in-depth analysis showed that MS-202 was the best-performing molecule among the optimized variants. Although MS-200, MS-201, and MS-202 had similar dimer formation rates at 40°C, MS-202 showed better resistance to subvisible particle formation over a 13-week period.

[0193] Antibody production

[0194] Antibody material was cloned and produced as previously described (Durocher, Y., Perret, S., & Kamen, A. (2002). Nucleic Acids Research, 30(2), E9). bNAb antibody material was produced by transient expression in two suspension cell lines: human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO). pTT5 mammalian expression vectors containing the coding regions for the light chain (LC) or heavy chain (HC) were co-transfected into HEK293 cells using polyethyleneimine (PEI) at a viable cell density (VCD) of 1*10^6 cells / mL (Durocher, Perret, & Kamen, 2002), and then diluted twice with preheated culture medium to 1 / 5 the volume of the shake flask. Expression was continued for 5-7 days at 37°C, 5% CO2, and 85% humidity, with a shaker speed of 130 RPM and a 19 mm orbit. The ExpiCHO-S was essentially prepared as described by ThermoFisher (Cat. No. A29133, File Part No. A29518). TM "Maximum titer" method. pcDNA3.4 expression vectors containing LC or HC coding regions were co-transfected into CHO-S cells with 6*10^6 VCDs using expifectamine. Expression was continued for 12 days at 37°C, 5% CO2 and 85% humidity, with a shaker speed of 130RPM and a 19mm orbit. All clear supernatants were produced by centrifugation of the cells at 3000g for 20 minutes and subsequent 0.22μm filtration. Antibodies were purified from the clear supernatant using Mab Select SuRe Protein A resin. A sodium phosphate, sodium chloride buffer system with arginine wash and pH 3.5 acetate elution was used. The Protein A eluate was neutralized with tris and the buffer exchanged to 20mM sodium phosphate, 150mM NaCl, pH 7.4.

[0195] Neutralization assay

[0196] Virus neutralization was assessed in TZM.b1 cells using a luciferase-based assay (J Virol 79(16): 10108-10125). The HIV-1 pseudoviruses tested primarily contained tier-2 and tier-3 viruses (Journal of Virology 84(3): 1439-1452). Virus neutralization was assessed in wild-type cells or HEK 293S GnTI cells treated with 25 μM kifunensine (Enzo Life Sciences). - / - Nonlinear regression analysis was used to calculate the concentration at which half-maximal inhibition was observed (IC 50 Values). Neutralizing activity was also assessed by a previously characterized PBMC-based assay using infection with major HIV-1 variants (n=95) isolated from clade B-infected donors with known seroconversion dates between 1985 and 1989 ("historical seroconverters", n=14) or between 2003 and 2006 ("contemporary seroconverters", n=21) (Journal of Virology 85(14):7236-7245; Nat Med 16(9):995-997). Neutralizing activity of each antibody was calculated using GraphPad Prism software (v5.0b) as the area under the best fit curve, which was fitted to an IC range of 0.001 to 50 μg / ml. 50 The value is the proportion of viruses neutralized.

[0197] HP-SEC

[0198] High performance size exclusion chromatography (" HP-SEC ") separates proteins based on the differences in their hydrodynamic volume. Molecules with larger hydrodynamic protein volumes elute earlier than molecules with smaller volumes. Undiluted samples were loaded onto a Waters XBridge Protein BEH SEC 200A column (3.5 μm, 7.8x300mm) and isolated isocratically using a running buffer of 100mM sodium phosphate, 250mM sodium chloride, pH 6.8, and the eluent was monitored by UV absorbance at 280nm. Purity was determined by calculating the percentage of each separated component relative to the total integrated area.

[0199] DSF

[0200] The DSF technique consists of measuring the fluorescence intensity of a hydrophobic probe at gradually increasing temperatures to determine the transition temperature and exposure of the hydrophobic region of the protein. The measurement results of this technique (expressed as transition temperature) correlate well with the data obtained from differential scanning calorimetry (DSC). DSF is a high-throughput technique for evaluating the relative thermodynamic stability of proteins and can be used as a tool for selecting candidates with favorable stability properties by ranking the results. Thermal transition temperatures were measured by DSF according to a previously described method (Feng H, et al. J Pharm Sci, 2010; 99: 4, 1707-1720). The analysis was performed in PBS buffer (20 mM sodium phosphate and 150 mM sodium chloride pH 7.1) with a final protein concentration of 0.15 mg / mL and a final Sypro Orange concentration of 3X. The protein and Sypro Orange were mixed in a 96-well PCR plate at a volume ratio of 1: 1 and analyzed using a Roche Light Cycler 480 instrument equipped with thermal shift analysis software. Thermal curves were generated by heating the sample from 20°C to 95°C at a ramp rate of 4.4°C / s with 10 acquisitions per°C, Ex = 465 nm Em = 580 nm. The first derivative of the melting curve was used to determine the transition temperature and shoulder score.

[0201] Low pH stability

[0202] The pH of a 1 mg / mL protein sample in 20 mM PBS was lowered to approximately pH 3.3 using 2 M acetic acid. After incubation for 30 minutes, the sample was neutralized to approximately pH 5 using 2 M Tris base. High molecular weight species in the sample were measured using the SE-HPLC method in duplicate. As a control, the protein sample was supplemented with the same volume of PBS as 2 M acetic acid and 2 M Tris base, and high molecular weight species were measured.

[0203] Relative solubility

[0204] Solubility was assessed according to previously described methods (Vishal M. Toprani, Sangeeta B. Joshi, Lisa A. Kueltzo, Richard M. Schwartz, C. Russell Middaugh, David B. Volkin). The micro-polyethylene glycol precipitation assay was used as a relative solubility screening tool for monoclonal antibody design and formulation development (J. Pharm. Sci 2016;105:8:2319-2327). The assay was performed in PBS buffer (20 mM sodium phosphate and 150 mM sodium chloride, pH 7.1) at a final PEG 10,000 concentration of 7.9%. 1 mg / mL of protein was diluted in PEG solution at a 1:4 ratio and incubated overnight at room temperature in 96-well 0.22 μm filter plates. After PEG incubation, the sample was passed through the filter by centrifugation, and the remaining soluble protein was measured by protein A titer assay.

[0205] Chemical unfolding

[0206] Liquid handling robot (liquid handling robot) is used to prepare 32 GND concentrations of 0 to 6M guanidine hydrochloride (GND) in PBS.Then, the protein sample of 1mg / mL in 20mM PBS is transferred to each GND concentration, to reach the final protein concentration of 0.05mg / mL. After incubation for 24 hours, sample (excitation: 280nm, emission: 300-450nm) is measured on SpectraMax M5 plate reader. By deducting a small amount of total intensity (for replacing the signal owing to scattering) measured between 300 and 320nm, the fluorescence intensity measured at 373nm is corrected for scattering and stray light, and then evaluated (ratioed) with the total intensity measured between 320 and 440nm to correct for total intensity fluctuation.Then, by mapping each corrected intensity relative to GND concentration to generate a chemical unfolding curve. Curve deflection has been calculated, and the curve deflection of each protein sample in this curve has been reported. Samples were measured in triplicate.

[0207] Subvisible particle analysis

[0208] Subvisible particles were measured using a Flowcam 8100 benchtop microfluidic imaging system equipped with an 80 μm flow cell and a 10X magnification lens, controlled by Visual Spreadsheet software. Samples were equilibrated to room temperature and gently swirled for thorough mixing. A single reading of 100 μl was collected for each sample, and the total concentration of particles larger than 2 μm was recorded.

[0209] Example 3

[0210] Characterization of the formation of oligomeric species and HMWs of the 10-1074 variant during virus inactivation and purification steps

[0211] Figure 1 shows the characterization of anti-HIV antibody 10-1074 variants MS-194 (Figure 1A) and MS-203 (Figure 1B) by high performance size exclusion chromatography ("HP-SEC") before and after virus neutralization. The arrows indicate the peaks in the HP-SEC spectra corresponding to oligomeric species formed during the virus inactivation process. Figure 2 shows the quantification of the degree of aggregation represented by the levels of high molecular weight ("HMW") and oligomeric species after each purification step of the 10-1074 antibody variants MS-194, MS-200, MS-201, and MS-203.

[0212] ExpiCHO-S was used essentially as described by ThermoFisher (Cat. No. A29133, File Part No. A29518). TM The "maximum titer" approach was used to produce molecules MS-194, MS-200, MS-201, and MS-203. Using expifectamine, pcDNA3.4 expression vectors containing either the light or heavy chain coding regions were co-transfected into 6 x 10^6 VCD CHO-S cells. Expression was continued for 12 days at 32°C, 5% CO2, and 85% humidity, on a shaker at 130 RPM and a 19 mm orbital. All clarified supernatants were obtained by centrifugation of the cells at 3000 g for 20 minutes and subsequent 0.22 μm filtration.

[0213] The antibody was purified from the clarified supernatant using MabSelect SuRe Protein A resin. Balanced with Tris and sodium chloride buffer. After loading, the column was washed with Tris buffer containing 0.5M sodium chloride. The bound mAb was eluted with 0.1M acetate buffer at pH 3.6 and then neutralized. The stability of each molecule during the viral inactivation process was determined by titrating the eluate to pH 3.5, incubating for 1 hour, and then neutralizing with Tris buffer. After elution, the remaining Protein A eluate was also immediately neutralized with a tris buffer system. Further purification was completed by loading the neutralized eluate onto Fractogel SO3-cation exchange resin (EMD Millipore Corporation) and eluting with a sodium chloride gradient. The peak containing mAb was collected, concentrated to 20 mg / mL, and the buffer was exchanged to 10 mM acetate, 9% sucrose, pH 5.2.

[0214] HP-SEC analysis was used as previously described to determine the percentage high molecular weight and oligomers for each sample. As shown in Figures 1A and 1B and quantified in Figure 2, the MS-194 antibody showed a significant increase in oligomers during low pH viral inactivation, while molecules MS-200, MS-201, and MS-203 showed no increase in HMW or oligomer content during viral inactivation.

[0215] Example 4

[0216] Characterization of 10-1074 variant stability

[0217] Figure 3 shows the HMW levels of the 10-1074 antibody variants MS-194, MS-200, MS-201, and MS-203 during incubation at 40°C for up to 13 weeks. The figure demonstrates similar rates of dimer formation during incubation at 40°C for up to 13 weeks. Figure 4 shows the levels of subvisible particle formation of the 10-1074 antibody variants MS-194, MS-200, MS-201, and MS-203 during 6 and 13 weeks. The figure demonstrates that the extent of micronization of antibodies MS-200, MS-201, and MS-203 was much less than that of MS-194. After 6 weeks, MS-194 exhibited 6-fold more particles than MS-200, MS-201, and MS-203, while after 13 weeks, MS-200 and MS-203 exhibited approximately 2-fold fewer particles than MS-194, and MS-203 exhibited approximately 4-fold less particle formation.

[0218] The monoclonal antibodies MS-194, MS-200, MS-201 and MS-203, which were purified and buffer-exchanged by cation exchange chromatography as previously described, were buffer-exchanged into 20 mM acetate, 9% sucrose and concentrated to 100 mg / mL at a final pH of 5.2. 500 μL aliquots of each sample were placed in 4 mL Type I glass vials and sealed with rubber stoppers and aluminum crimp seals. The samples were incubated at 40°C for up to 13 weeks. Samples were removed at designated time points and the vials were then resealed and placed back in the incubator. As described above, the HMW percentage was determined using HP-SEC, and sub-visible particles were determined using a FlowCam instrument.

[0219] Example 5

[0220] Combination therapy with anti-HIV-1 antibodies

[0221] Although anti-HIV-1 antibodies constitute a potential alternative to ART5, treatment of viremic individuals with a single antibody can also lead to the emergence of resistant viral variants ((Caskey, M. et al. Nature 522, 487-491 (2015); Caskey, M. et al. Nat. Med. 23, 185-191 (2017); Lynch, RM et al. Sci. Transl. Med. 7, 319ra206 (2015). In addition, the combination of the first generation of anti-HIV-1 broadly neutralizing antibodies (bNAbs) had little measurable effect on infection. The present disclosure provides the results of a Phase 1b clinical trial (NCT02825797) in which a combination of two potent monoclonal anti-HIV-1 broadly neutralizing antibodies 3BNC117 and 10-1074 targeting different sites on the HIV-1 envelope spike was administered during an analytical treatment interruption (ATI) (Mendoza et al., Nature. 2018 Sep; 561(7724): 479-484; Bar-On et al., Nature Medicine 24: 1701-1707 (2018). Participants received three infusions of 30 mg / kg of each antibody at weeks 0, 3, and 6. Infusions of both antibodies were generally well tolerated. The nine individuals enrolled with antibody-sensitive latent viral pools remained suppressed for 15 to >30 weeks (median = 21 weeks). In the four individuals with dual antibody-sensitive virus, immunotherapy reduced HIV-1 viral load by an average of 2.05 log per milliliter. 10The researchers found that the combination of anti-HIV-1 monoclonal antibodies 3BNC117 and 10-1074 can maintain long-term suppression in individuals with antibody-sensitive viral pools in the absence of ART.

[0222] Study Design

[0223] An open-label, phase 1b study (http: / / www.clinicaltrials.gov; NCT02825797; EudraCT: 2016-002803-25) was conducted in HIV-1 infected participants who were virologically suppressed on antiretroviral therapy (ART) (Mendoza et al., Nature. 2018 Sep; 561(7724): 479-484; Bar-On et al., Nature Medicine 24: 1701-1707 (2018)). Study participants were enrolled sequentially according to eligibility criteria. Participants received 3BNC117 and 10-1074 intravenously at a dose of 30 mg / kg body weight of each antibody at weeks 0, 3, and 6, unless viral rebound occurred. ART was discontinued 2 days after the first infusion of antibody (day 2). Plasma HIV-1 viral RNA levels were monitored weekly and if viral load increased to ≥200 copies / ml or CD4 + ART was resumed when the T-cell count dropped to <350 cells / pl. The time of viral rebound was determined when the viral load was >200 copies / ml for the first time. After the first infusion, study participants were followed up for 30 weeks. Safety data were reported until the end of the study follow-up. All participants provided written informed consent before participating in the study, and the study was conducted in accordance with Good Clinical Practice (GCP). The protocol has been approved by the Federal Drug Administration (FDA), the Paul-Ehrlich-Institute in Germany, and the Institutional Review Boards (IRBs) of Rockefeller University and the University of Cologne.

[0224] Study participants

[0225] Study participants were recruited at Rockefeller University Hospital, New York, USA, and University Hospital Cologne, Cologne, Germany. Eligible participants were adults aged 18 to 65 years, infected with HIV-1, receiving ART for at least 24 months, with plasma HIV-1 RNA levels <50 copies / ml for at least 18 months (a single viral blip >50 but <500 copies / ml during this 18-month period was permitted), plasma HIV-1 RNA levels <20 copies / ml at the screening visit, and current CD4 + T cell counts were >500 cells / pl. In addition, participants were prescreened for sensitivity of latent provirus to 3BNC117 and 10-1074 by bulk PBMC viral growth culture as described below. Sensitivity was defined as IC50 <2 pg / ml for both 3BNC117 and 10-1074 against growing virus. Due to the longer half-life of NNRTIs, participants on NNRTI-based ART regimens were switched to an integrase inhibitor-based regimen (dolutegravir plus tenofovir disoproxil fumarate / emtricitabine) 4 weeks before treatment interruption. Exclusion criteria included reported CD4 + T-cell nadir <200 cells / pl, concurrent hepatitis B or C infection, previous exposure to any class of monoclonal antibodies, clinically relevant physical findings, medical conditions, or laboratory abnormalities, and pregnancy or lactation.

[0226] Research Process

[0227] 3BNC117 and 10-1074 were administered intravenously at a dose level of 30 mg / kg (Mendoza et al., Nature. 2018 Sep;561(7724):479-484; Bar-On et al., Nature Medicine 24:1701-1707(2018)). Appropriate 3BNC117 and 10-1074 stock volumes were calculated based on body weight and diluted in sterile saline to a total volume of 250 ml for each antibody. Monoclonal antibody infusions were administered sequentially and intravenously over 60 minutes. After the last antibody infusion, study participants were observed for one hour at the Rockefeller University Hospital or the University Hospital of Cologne. Participants returned for weekly follow-up visits during the ATI for safety assessments, which included physical examinations and measurement of clinical laboratory parameters such as hematology, chemistry, urinalysis, and pregnancy tests (for women). Plasma HIV-1 RNA levels were monitored weekly during the ATI, and CD4 was measured every 1 to 2 weeks. + T cell counts. After restarting ART, participants returned for follow-up every 2 weeks until viral resuppression was achieved and every 8 weeks thereafter. Study investigators evaluated and scored adverse events according to the DAIDS AE grading scale (version 2.0, November 2014), and causality was determined. Leukapheresis was performed at the Rockefeller University Hospital or the University Hospital of Cologne at weeks -2 and 12. Blood samples were collected multiple times before and after 3BNC117 and 10-1074 infusion. Samples were processed within 4 hours of collection, and serum and plasma samples were stored at -80°C. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. The absolute number of PBMCs was determined by an automated cell counter (Vi-Cell XR; Beckman Coulter) or manually, and the cells were cryopreserved in fetal bovine serum plus 10% DMSO.

[0228] Plasma HIV-1 RNA levels

[0229] HIV-1 RNA levels in plasma were measured at screening, at week -2, on day 0 (before infusion), weekly during ATI, and every two to eight weeks after viral rebound. HIV-1 RNA levels were determined using the Roche COBAS AmpliPrep / COBASTaqMan HIV-1 assay (version 2.0) or the Roche COBAS HIV-1 quantitative nucleic acid test (COBAS 6800), which can detect viral loads at 2 × 10 1 to 1×10 7HIV-1 RNA was quantified in the range of 10 copies / ml. These assays were performed at LabCorp or the University Hospital of Cologne.

[0230] CD4+ T cells

[0231] CD4 was determined by clinical flow cytometry at LabCorp or the University Hospital of Cologne at screening, at week 0 (before infusion), and weekly at weeks 2, 3, 5, 6, 8, 10, and thereafter (participants remained ART-free). + T cell count.

[0232] Determination of baseline neutralizing antibody activity

[0233] Purified IgG obtained prior to antibody infusion (Protein G Sepharose 4 Fast Flow, GE LifeSciences) was tested against a panel of 12 HIV-1 pseudoviruses as described previously (Schoofs T et al. Science 352, 997-1001 (2016)).

[0234] Measurement of 3BNC117 and 10-1074 serum levels

[0235] Blood samples were collected before and at the end of each 3BNC117 infusion and at the end of each 10-1074 infusion at Weeks 0, 3, and 6, and weekly during the ATI until Week 30. Serum levels of 3BNC117 and 10-1074 were determined by TZM-bl assay and ELISA on samples obtained before and after each antibody infusion, and approximately every three weeks during follow-up and in the event of viral rebound.

[0236] Serum concentrations of 3BNC117 and 10-1074 were measured using a validated sandwich ELISA. High-binding polystyrene plates were coated with 4 μg / ml of an anti-idiotypic antibody specifically recognizing 3BNC117 (anti-ID 1F1-2E3 mAb) or 2 μg / ml of an anti-idiotypic antibody specifically recognizing 10-1074 (anti-ID 3A1-4E11 mAb) and incubated overnight at 2-8°C. After washing, the plates were blocked with PBS containing 5% Milk Blotto (w / v), 5% NGS (v / v), and 0.05% Tween 20 (v / v). Serum samples, QCs, and standards were added (at a minimum dilution of 1:50 in PBS containing 5% Milk Blotto (w / v), 5% NGS (v / v), and 0.05% Tween 20 (v / v)) and incubated at room temperature. 3BNC117 or 10-1074 were detected using a horseradish peroxidase (HRP)-conjugated mouse anti-human IgG kappa chain specific antibody (Abcam) for 3BNC117 or an HRP-conjugated goat anti-human IgG Fc specific antibody (Jackson ImmunoResearch) for 10-1074, along with the HRP substrate tetramethylbenzidine. The concentrations of 3BNC117 and 10-1074 were then calculated using a 5-PL curve fitting algorithm (Softmax Pro, v5.4.5) based on the standard curves of 3BNC117 or 10-1074 run on the same plate. Standard curves and positive controls were created based on the 3BNC117 and 10-1074 drugs used in clinical studies. Capture anti-idiotypic mAbs were produced using a stable hybridoma cell line (Duke Protein Production Facility). The lower limit of 3BNC117ELISA quantification is 0.78 μ g / ml, and is 0.41 μ g / ml for 10-1074ELISA. Determine that the detection lower limit of 3BNC117 and 10-1074ELISA in HIV-1 seropositive serum is 0.51 μ g / ml and 0.14 μ g / ml respectively. For detectable (i.e. mAb positive) but lower than the value of quantitative lower limit, the value of 3BNC117 and 10-1074ELISA is reported as <0.78 μ g / ml and <0.41 μ g / ml respectively. If the baseline sample has measurable antibody level by corresponding determination at day 0, the measured background antibody level is subtracted from subsequent results. In addition, samples measuring antibody level within 3 times of background are excluded from the analysis of PK parameters.

[0237] As previously described (Sarzotti-Kelsoe M et al. J Immunol Methods 409, 131-146 (2014)), serum concentrations of active 3BNC117 and 10-1074 were also measured in TZM-bl cells using a validated luciferase-based neutralization assay. Briefly, serum samples were tested using a 1:20 primary dilution and a 5-fold titration series against HIV-1 Env pseudoviruses Q769.d22 and X2088_c9, both of which were highly sensitive to neutralization by 3BNC117 and 10-1074, respectively, while being completely resistant to the other administered antibodies. For the post-infusion time point of 10-1074, serum samples were also tested against the less sensitive strain Du422 when the serum ID50 titer against X2088_c9 was >100,000. To generate the standard curve, 3BNC117 and 10-1074 clinical drugs were included in each assay setup using an initial concentration of 10 μg / ml and a 5-fold titration series. The serum concentration of 3BNC117 and 10-1074 for each sample was calculated as follows: serum ID50 titer (dilution) × 3BNC117 IC50 Or 10-1074IC50 titer (μg / ml) = 3BNC117 or 10-1074 serum concentration (μg / m1). Env pseudovirus (SG3ΔEnv / K101P.Q148H.Y181C) was produced using an anti-ART backbone vector that reduces the background inhibitory activity of antiretroviral drugs when present in serum samples. A virus pseudotyped with murine leukemia virus (MuLV) envelope protein was used as a negative control. If non-specific activity against MuLV was detected (ID50>20; 9246, week 30; 9248, baseline, d0, wk 18), the antibody concentration was calculated using serum ID80 titer and monoclonal antibody IC80. All assays were performed in a GCLP-compliant laboratory.

[0238] Prescreening of large PBMC cultures

[0239] To test the sensitivity of HIV-1 strains to 3BNC117 and 10-1074, isolated CD4 + T cells with MOLT-4 / CCR-5 or CD8 + Large-scale viral growth cultures were co-cultured with donor lymphoblasts of T cells. PBMCs for prescreening were obtained up to 72 weeks (range, 54-505 days) prior to enrollment, according to separate protocols approved by the IRBs of Rockefeller University and the University of Cologne. Sensitivity was determined by the TZM-b1 neutralization assay, as described below. Culture supernatants with an IC50 of <2 μg / ml were considered sensitive.

[0240] Quantitative and Qualitative Viral Growth Assay (Q2VOA)

[0241] Quantitative and qualitative viral growth assays (Q 2 VOA). Briefly, the isolated CD4 + T cells were activated with 1 μg / ml phytohemagglutinin (Life Technologies) and 100 U / ml IL-2 (Peprotech) and incubated with 1×10 6 6 × 10 irradiated PBMCs were co-cultured in 24-well plates. A total of 6 × 10 7 -6.2×10 8 After 24 hours, PHA was removed and 0.1×10 6 MOLT-4 / CCR5 cells were plated. Cultures were maintained for 2 weeks, with half of the MOLT-4 / CCR5 cells plated 7 days after initiation of culture and every other day thereafter. Positive wells were detected by measuring p24 using an ELISA. The frequency of latently infected cells was calculated using the infectious units per million (IUPM) algorithm developed by the Siliciano laboratory (http: / / silicianolab.johnshopkins.edu).

[0242] Rebound growth culture

[0243] Strictly follow Q 2 CD4 isolated from PBMCs from the rebound time point were cultured at limiting dilution as described in VOA. + T cells. 6 CD4 + T cells 0.5×10 6 The concentration of beads was 20 U / ml and CD4 T cells were activated using T cell activation beads (Miltenyi) and 20 U / ml IL-2. + T cells. Rebound growth was performed using PBMCs from the highest viral load samples (usually repeated measurements of ≥200 copies / ml). Viruses whose sequences matched the SGA env sequences and were therefore identical to those present in the plasma (as opposed to latent pool viruses of potential reactivated PBMC origin) were selected for neutralization testing.

[0244] Virus sensitivity test

[0245] Cells from p24-positive bulk PBMC cultures, rebound PBMC outgrowth cultures, and Q cells were tested by TZM-bl neutralization assay as described previously (Sarzotti-Kelsoe M et al. J Immunol Methods 409, 131-146 (2014)). 2 Sensitivity of supernatants from VOA wells to 3BNC117 and 10-1074.

[0246] Sequencing

[0247] HIV-1 RNA extraction and single genome amplification were performed as previously described (Salazar-Gonzalez JF et al. J Virol 82, 3952-3970 (2008)). Briefly, HIV-1 RNA was extracted from plasma samples or QIAGEN using the MinElute Virus Spin kit (Qiagen). 2 HIV-1 RNA was extracted from the viral supernatant of VOA origin, and the first-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). The cDNA synthesis of plasma-derived HIV-1 RNA was performed using the antisense primer envB3out Fidelity Platinum Taq (Invitrogen) and running at 94°C for 2 minutes; 94°C for 15s, 55°C for 30s and 68°C for 4 minutes, 35 cycles; and 68°C for 15 minutes. The second round of PCR used 1 μl of the first PCR product as a template and High Fidelity Platinum Taq and was performed at 94°C for 2 minutes; 94°C for 15s, 55°C for 30s and 68°C for 4 minutes, 45 cycles; and 68°C for 15 minutes. Q was performed using the antisense primer R3B6R 2 cDNA synthesis of VOA-derived HIV-1 RNA.

[0248] Study Results

[0249] Combined bNab infusion was well tolerated

[0250] To evaluate the effect of the combination of 3BNC117 and 10-1074 in maintaining HIV-1 suppression during ATI, a Phase 1b clinical trial was conducted (Figure 5A) (Mendoza et al., Nature. 2018 September; 561(7724): 479-484). Using the TZM-b1 neutralization assay, individuals infected with HIV-1 on ART were pre-screened for sensitivity to 3BNC117 and 10-1074 against a large number of viruses derived from growing cultures. Consistent with previous results, 64% and 71% of the growing viruses were sensitive to 3BNC117 and 10-1074, respectively, and 48% were sensitive to both (IC50 ≤ 2 μg / ml).

[0251] Study eligibility criteria included at least 24 months of ART, plasma HIV-1 RNA levels <50 copies / ml for at least 18 months (one intermittent viremia <500 copies / ml was allowed) and <20 copies / ml at screening, and CD4 + T-cell counts >500 cells / μl. Enrolled participants received three infusions of 3BNC117 + 10-1074 at 30 mg / kg each every 3 weeks starting 2 days before treatment interruption (Figure 5A). Individuals whose regimen contained a non-nucleoside reverse transcriptase inhibitor were switched to an integrase inhibitor-based regimen 4 weeks before stopping ART (Figure 6A). Viral load and CD4 were monitored every 1 to 2 weeks. + T cell counts were performed. If viremia >200 copies / ml was confirmed, ART was restarted and antibody infusions were stopped. The time to viral rebound was defined as the first of two consecutive viral loads >200 copies / ml. Fifteen individuals were enrolled, but four of them showed viral loads >20 copies / ml two weeks before or at the time of the first bNAb infusion and were excluded from the efficacy analysis.

[0252] The antibody infusions were generally safe and well tolerated, with no serious adverse events or antibody-related adverse events reported, except for two participants who experienced mild fatigue. + T-cell counts were 685 and 559 cells / μl, respectively (Figure 6B). Reinitiating ART after viral rebound resulted in resuppression of viremia. In conclusion, the combination of 3BNC117 and 10-1074 was generally safe and well tolerated.

[0253] Combination with bNAb to maintain viral suppression

[0254] For 11 individuals with complete viral suppression (HIV-1 RNA <20 copies / m1) during the screening period and on day 0, combined antibody therapy was associated with maintenance of viral suppression for 5 to >30 weeks (Figures 5B and 5C) (Mendoza et al., Nature. 2018 September; 561(7724): 479-484). The median time to rebound was 21 weeks, compared to 2.3 weeks for historical controls participating in non-interventional ATI studies and 6-10 weeks for 3BNC117 monotherapy (Figure 5C). In summary, 9 of the 11 participants maintained viral suppression within 15 weeks, while 2 participants rebounded at weeks 5 and 7 (Figures 5B and 5C).

[0255] Quantitative and qualitative viral growth assays (Q 2 VOA) retrospectively analyzed the replication-competent latent virus pool in all individuals. Phylogenetic analysis showed that trial participants were infected with epidemiologically distinct evolutionary clade B viruses. 2 VOA analysis showed that the pre-infusion latent pools in the two individuals 9245 and 9251 who rebounded early carried viruses against 10-1074 or 3BNC117, respectively (Fig. 7). Therefore, these two individuals effectively underwent antibody monotherapy because there was pre-existing resistance to one of the two bNAbs in the pool. Consistent with this idea, the rebound delay in these two participants was within the expected range of antibody monotherapy (Fig. 5C). In addition, all four individuals excluded from the analysis due to incomplete viral suppression showed pre-existing resistance or viruses that were not fully neutralized by one or both antibodies, and these individuals rebounded before week 12.

[0256] In order to check the virus produced in the early rebound individual, single genome analysis (SGA) was carried out to rebound plasma. The bNAb sensitivity of the pseudovirus constructed from plasma SGA was detected in TZM-b1 determination. In addition to the pre-existing sequence (N332T+S334N, Fig. 7 A) related to resistance in the 10-1074 target site, the rebound virus in 9245 also carried an extended V5 loop and a potential N-linked glycosylation site that can interfere with 3BNC117 binding. In contrast, the genetic signature related to resistance to 3BNC117 was found in the pre-infusion pool of 9251, and it was accompanied by a mutation (S334N, Fig. 7 A) in the 10-1074 target site in the rebound virus. For two individuals, the resistance (Fig. 7 B and 7C) of the rebound virus to two antibodies was confirmed by TZM-b1 neutralization assay. Therefore, a large amount of growth cultures for screening failed to detect pre-existing resistance in the pools of 2 of the 11 individuals studied. This result is not surprising given that bulk cultures are dominated by a limited number of rapidly growing viral species that may not represent the diversity of the latent pool.

[0257] Similarly, participant 91C33, who did not respond to the antibody infusion, had pre-existing circulating viruses that were resistant to both antibodies (Bar-On et al., Nature Medicine 24:1701-1707 (2018)). These viruses carried mutations at the 3BNC117 contact site (N280S and A281H) and the 10-1074 contact site (N332T and S334N). Two individuals, 91C35 and 9341, responded to the antibody therapy with a reduction in viremia of -1.58 and -1.32 log, respectively. 10 copies / ml, but HIV-1 RNA levels returned to baseline within 3 and 4 weeks, respectively. 91C35 was found to have pre-infusion circulating virus with reduced sensitivity to 3BNC117 and to carry a mutation (A281T) in a CD4 contact residue associated with viral escape from 3BNC11720. + Pre-infusion virus from T cell growth cultures showed a 1.3 log higher geometric mean IC80 than all other enrolled viremic individuals 10 The 10-1074 IC80 was significantly higher than that of the 10-1074 IC80. In both cases, the rebound viruses were resistant to both antibodies and carried a mutation that resulted in the loss of a potential N-linked glycosylation site at position 332, which is critical for 10-1074 binding. Furthermore, rebound viruses from 91C35 and 9341 contained the G471E and N276D mutations, respectively, which are associated with increased resistance to 3BNC117. These mutations were not found in the pre-infusion circulating viruses described above or in the other 113 pre-infusion env sequences analyzed from these two participants. Therefore, 91C35 and 9341 were infected with viruses with reduced sensitivity to one of the two antibodies, and the magnitude of the decline in viremia and the time to return to baseline viremia were similar to those in individuals receiving antibody monotherapy. In conclusion, bulk growth cultures used for initial screening failed to detect partial or complete pre-existing resistance to one or both antibodies in three of the seven individuals studied.

[0258] The remaining four individuals showed no detectable pre-existing resistant virus in circulation and experienced significantly suppressed viremia until day 94 after the first antibody infusion, with a mean maximum viral load decrease of -2.05 log per milliliter. 10copies per milliliter (Bar-On et al., Nature Medicine 24:1701-1707 (2018)). The individual with the highest initial viral load in this group (97,800 copies per milliliter; patient 9343) was the first to rebound at 8 weeks. The two individuals with the lowest initial viral load, 91C22 and 9342 (750 and 2,550 copies per milliliter, respectively), showed inhibition close to or below the limit of detection at 12 weeks and 16 weeks, respectively. Finally, viremia in participant 91C34 decreased for 12 weeks, but it never fell below 810 copies per milliliter. Despite persistent viremia, this individual did not develop resistance to either antibody as long as bNAb serum levels were above 10 μg / ml. In three of the four initially sensitive individuals, rebound viremia was associated with the emergence of virus resistant to 10-1074, but these individuals remained sensitive to 3BNC117. This is consistent with the relatively short half-life of 3BNC117, which means that participants were effectively exposed to 10-1074 monotherapy at the end of the observation period. Consistent with the increased resistance to 10-1074, rebound viruses carry mutations in the contact sites of 10-1074. In contrast, there was no accumulation of new mutations at the 3BNC117 contact sites. Participant 91C22, who had the lowest initial viral load, only returned to baseline viremia after both antibodies were below the detection limit, and rebound viruses remained sensitive to both antibodies. Overall, the four participants who were initially sensitive to these two antibodies did not develop new resistance to 3BNC117 during the cumulative observation period of more than one year (56 weeks), although residual viremia was observed in three of the participants, and recombination events frequently occurred between circulating viruses.

[0259] Among the seven individuals who had no detectable resistant virus in the pre-infusion latent pool and rebounded during the study, the median time to rebound was also 21 weeks, and was different from the 6-10 weeks for 3BNC117 monotherapy (Figure 5C) (Mendoza et al., Nature. 2018 September; 561(7724): 479-484). In these participants, viral suppression was maintained for 15 to 26 weeks after ART cessation. The remaining two participants (9254 and 9255) completed the study follow-up within 30 weeks and did not experience rebound. It is worth noting that viral rebound never occurred when the concentrations of both antibodies administered exceeded 10 μg / ml. The average 3BNC117 serum concentration (determined by TZM-b1 assay) at the time of rebound for sensitive individuals who rebounded during the study follow-up was 1.9 μg / ml (Figure 5B). In contrast, the average serum concentration of 10-1074 at the time of rebound was 14.8 μg / ml (Figure 5B). The difference in antibody concentrations at rebound is consistent with the longer half-life of 10-1074, which resulted in a period of 10-1074 monotherapy (Figure 5B). Finally, these nine individuals showed little or no pre-existing neutralizing antibodies against the viral diagnostic panel before bNAb infusion.

[0260] Rebound and latent viruses

[0261] To explore the relationship between rebound viruses and the circulating latent pool, env sequences obtained from plasma rebound viruses by SGA were compared with those obtained by Q 2 Sequences obtained from the pre-infusion and week 12 samples were compared with those obtained from the VOA. In addition, the susceptibility of rebound virus and / or pseudovirus to 3BNC117 and 10-1074 was measured by the TZM-b1 neutralization assay (Figures 7B and 7C). A total of 154 viral env sequences obtained by plasma SGA were analyzed and compared with those obtained by Q 2 VOA compared 408 sequences obtained from the latent pool. Although rebound viruses and pool viruses in each individual clustered together, no identical sequences were found between the two compartments of any individual studied (Figures 8 and 9A). The differences may be explained by different requirements for HIV-1 reactivation in vivo and in vitro, grouping of pool viruses, HIV-1 mutations during the experiment, and / or viral recombination in some individuals. Whether bNAb therapy affects the selection of recombination events remains to be determined.

[0262] Similar to 3BNC117 monotherapy, the vast majority of rebound viruses clustered in low-diversity lineages, consistent with the expansion of 1-2 recurrent viruses (Figure 8). In contrast, in the absence of antibody therapy, rebound viruses were consistently polyclonal during ATI. Thus, antibodies limit the growth of latent viruses in vivo.

[0263] When the average 3BNC117 and 10-1074 concentrations were 1.9 and 14.8 μg / ml, respectively, emerging viruses that rebounded in six of seven individuals carried resistance-associated mutations at the 10-1074 target site (Figures 5B and 8). Consistent with the sequence data, these rebound viruses were generally resistant to 10-1074 (as determined by TZM-bl neutralization assay) but remained sensitive to 3BNC117 (Figures 7B and 7C). The level of sensitivity of these emerging viruses to 3BNC117 was similar to that found in the pooled viruses from each individual (Figure 7B). One individual, 9244, showed rebound viruses that remained sensitive to both antibodies in the TZM-bl neutralization assay. Rebound occurred when the serum concentrations of 3BNC117 and 10-1074 in this individual were undetectable and 11.6 μg / ml, respectively (Figure 5B). The sensitivity of the plasma rebound virus was similar to that of the latent pre-infusion virus and the week 12 virus obtained from the virus growth culture (Figures 7B and 7C). Therefore, despite prolonged exposure to both antibodies, this individual did not develop resistance to either of the two antibodies. In summary, none of the nine individuals with pre-infusion pools containing virus sensitive to both antibodies developed dual resistance during the observation period.

[0264] Lurking Pool

[0265] To determine whether the circulating pool changed during the observation period, Q was compared between the 8 of the 9 individuals who remained suppressed for at least 12 weeks and the 12 weeks after ATI initiation. 2 VOA assay results (Figure 9). Similar to previous reports, the 2 63% of all viruses obtained by VOA belonged to expanded clones. Comparison of env sequences of emerging viruses in growing cultures showed that 60% of the sequences could be found at both time points. However, there were many examples of clones appearing or disappearing between these time points, and some of these changes were significant. To determine the number of infectious units per million (IUPM, http: / / silicianolab.johnshopkins.edu / ), the env sequences were obtained by Q 2 VOA was measured for each individual at each time point at 6.0×10 7 -6.2×10 8 CD4 + T cells (Figure 9B). For any individual, the difference between the two time points was never greater than 6.5-fold, and the two time points were not statistically different (P = 0.078). Moreover, rebound time was not directly correlated with IUPM. Additional time points will be needed to calculate the half-life of the pool in individuals receiving immunotherapy.

[0266] discuss

[0267] In animal models and humans, first-generation anti-HIV-1 bNAbs generally fail to effectively suppress viremia, leading to the conclusion that this approach should not be pursued. In humans infected with HIV-1, bNAb monotherapy with either 3BNC117 or VRC01 is insufficient to maintain control during ATI. In contrast, the combination of 3BNC117 and 10-1074 is sufficient to maintain viral suppression in susceptible individuals when serum concentrations of both antibodies remain above a certain level, for example, above 10 μg / ml. When 3BNC117 levels fall below 10 μg / ml, effectively leading to 10-1074 monotherapy, rebound occurs, at which point nearly all individuals rapidly escape due to mutations within the 10-1074 contact site. Nine individuals infected with different viruses were observed to be unable to develop dual-resistant virus within a median of 21 weeks, indicating that viral replication is severely restricted by this antibody combination.

[0268] In human studies, 3BNC117 monotherapy was associated with enhanced humoral immunity and accelerated clearance of HIV-1 infected cells. Furthermore, when administered early to SHIVAD8-infected macaques, combined 3BNC117 + 10-1074 immunotherapy induced host CD8 + T cell responses, which helped control viremia in nearly 50% of the animals. However, virus-specific CD8 T cells, which are responsible for controlling viremia in these macaques, were not detected in the circulation. + T cells, and only in CD8 + T cell contribution to viral suppression was documented only after T cell depletion. In most control macaques, full viral suppression was established only after rebound viremia following antibody clearance.

[0269] Two individuals in this study, 9254 and 9255, remained suppressed for more than 30 weeks after ATI. Neither of them had detectable ART levels in their blood, nor did they carry the B*27 and B*57 HLA alleles most frequently associated with elite control (Walker BD & Yu XG. Nat Rev Immunol 13, 487-498 (2013)). The first, 9254, reported starting ART within 4-5 months of possible exposure to the virus with an initial viral load of 860,000 copies / ml. Despite relatively early initiation of therapy and 21 years of excellent virological control on therapy, this individual had an IUPM of 0.68 at the 12-week time point (by Q 2VOA). The second individual, 9255, showed several episodes of intermittent viremia that began to spontaneously control 15 weeks after ATI, when antibody levels declined. This individual had been infected for at least 7 months before starting ART, with an initial viral load of 85,800 copies / ml and an IUPM of 1.4 at the 12-week time point. A small proportion of individuals on ART show spontaneous, prolonged virologic control after stopping ART, and this number appears to increase when ART treatment is started during the acute phase of infection.

[0270] A large fraction of the circulating latent pool consists of expanded clones of infected T cells. These T cell clones appear to be dynamic because circulating latently infected CD4 + The specific contribution of individual clones of T cells to the pool in individuals receiving ART fluctuates over time. Individuals who maintain viral suppression with antibody therapy appear to show similar fluctuations in pool clones, which do not appear to be related to antibody sensitivity. Whether the apparent differences observed in the pool during immunotherapy will lead to changes in the pool half-life cannot be determined from the available data and will require evaluation of the pool in additional individuals at multiple time points over an extended observation period.

[0271] Individuals with viruses susceptible to 3BNC117 and 10-1074 maintained viral suppression for a median of nearly 4 months after the last administration of antibodies during the ATI period. In macaques, the efficacy of anti-HIV-1 antibodies is directly related to their half-life, which can be extended by mutations that enhance the interaction of the Fc domain with the neonatal Fc receptor. Such mutations can extend the half-life of antibodies in humans by 2-4 fold. The data suggest that a single administration of a bNAb combination with an extended half-life can maintain suppression for 6 to 12 months in individuals with susceptible virus.

[0272] Table 1: Residue numbering of anti-HIV antibody 10-1074 variant MS-194

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] Table 4: Amino acid sequences of the CDR regions of the 10-1074 antibody variants

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425] Table 7: Nucleic acid sequences of CDR regions of 10-1074 antibody variants

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448] Table 8: Methods for characterizing anti-HIV antibody 10-1074 variants

[0449]

[0450] Table 9: Molecular and biophysical analysis of 10-1074 antibody variants (Round 1)

[0451]

[0452] Table 10: Neutralization analysis against 10-1074 sensitive virus panel in molecular panel and TZM.b1 cells (Round 1)

[0453]

[0454]

[0455] Table 11: Reasons for inclusion or exclusion of variants based on neutralization activity and biophysical analysis (Round 1)

[0456]

[0457]

[0458] Table 12: Molecular panel of anti-HIV antibody 10-1074 variants (round 2)

[0459]

[0460]

[0461] Table 13: Molecular and biophysical analysis of 10-1074 antibody variants (Round 2)

[0462]

[0463]

[0464] Table 14: Additional biophysical characteristics of combinatorial variants (Round 2)

[0465]

[0466]

[0467]

[0468] Table 15: Neutralization analysis in TZM.bl cells of variants selected in round 2. Potency loss values ​​> 3 times the control value.

[0469]

[0470]

[0471] Table 15 - continued

[0472]

[0473]

[0474] Table 16: Reasons for exclusion of combinatorial variants based on biophysical analysis (Round 2)

[0475]

[0476]

[0477] The foregoing examples and description of the preferred embodiments should be considered as illustrative, rather than limiting, of the invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features described above may be utilized without departing from the invention as set forth in the claims. Such variations are not to be considered as departing from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated herein in their entirety. Sequence Listing <110> The Rockefeller University: Michel Nussenzweig, Randal R. Ketchem, Christine C. Siska, Alison J. Gillespie, Rutilio H. Clark, Bruce A. Kerwin <120> Anti-HIV antibody 10-1074 variant <130> 070413.20397 <150> 62 / 731,356 <151> 2018-09-14 <160> 753 <170> PatentIn version 3.5 <210> 1 <211> 233 <212> PRT <213> Homo sapiens <400> 1Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp AspSer Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 2 <211> 233 <212> PRT <213> Homo sapiens <400> 2 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 7580Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 3<211> 233<212> PRT<213> Homo sapiens<400> 3Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His ArgPro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 4<211> 233<212> PRT<213> 智人(Homo sapiens)<400> 4Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Pro Pro Leu Ser 脯氨酸-甘氨酸-谷氨酰胺-丙氨酸-脯氨酸-异亮氨酸-亮氨酸-亮氨酸-异亮氨酸-酪氨酸-天冬酰胺50 55 60天冬酰胺-谷氨酰胺-天冬氨酸-精氨酸-脯氨酸-丝氨酸-甘氨酸-异亮氨酸-脯氨酸-谷氨酸-精氨酸-苯丙氨酸-丝氨酸-甘氨酸-苏氨酸-脯氨酸65 70 75 80天冬氨酸-异亮氨酸-天冬酰胺-苯丙氨酸-甘氨酸-苏氨酸-精氨酸-丙氨酸-苏氨酸-亮氨酸-苏氨酸-异亮氨酸-丝氨酸-甘氨酸-缬氨酸-谷氨酸85 90 95丙氨酸-甘氨酸-天冬氨酸-谷氨酸-丙氨酸-天冬氨酸-酪氨酸-酪氨酸-半胱氨酸-组氨酸-甲硫氨酸-色氨酸-天冬氨酸-丝氨酸-精氨酸-丝氨酸100 105 110甘氨酸-苯丙氨酸-丝氨酸-色氨酸-丝氨酸-苯丙氨酸-甘氨酸-甘氨酸-丙氨酸-苏氨酸-精氨酸-亮氨酸-苏氨酸-缬氨酸-亮氨酸-甘氨酸115 120 125谷氨酰胺-脯氨酸-赖氨酸-丙氨酸-丙氨酸-脯氨酸-丝氨酸-缬氨酸-苏氨酸-亮氨酸-苯丙氨酸-脯氨酸-脯氨酸-丝氨酸-丝氨酸-谷氨酸130 135 140谷氨酸-亮氨酸-谷氨酰胺-丙氨酸-天冬酰胺-赖氨酸-丙氨酸-苏氨酸-亮氨酸-缬氨酸-半胱氨酸-亮氨酸-异亮氨酸-丝氨酸-天冬氨酸-苯丙氨酸145 150 155 160酪氨酸-脯氨酸-甘氨酸-丙氨酸-缬氨酸-苏氨酸-缬氨酸-丙氨酸-色氨酸-赖氨酸-丙氨酸-天冬氨酸-丝氨酸-丝氨酸-脯氨酸-缬氨酸165 170 175赖氨酸-丙氨酸-甘氨酸-缬氨酸-谷氨酸-苏氨酸-苏氨酸-苏氨酸-脯氨酸-丝氨酸-赖氨酸-谷氨酰胺-丝氨酸-天冬酰胺-天冬酰胺-赖氨酸180 185 190酪氨酸-丙氨酸-丙氨酸-丝氨酸-丝氨酸-酪氨酸-亮氨酸-丝氨酸-亮氨酸-苏氨酸-脯氨酸-谷氨酸-谷氨酰胺-色氨酸-赖氨酸-丝氨酸195 200 205组氨酸-精氨酸-丝氨酸-酪氨酸-丝氨酸-半胱氨酸-谷氨酰胺-缬氨酸-苏氨酸-组氨酸-谷氨酸-甘氨酸-丝氨酸-苏氨酸-缬氨酸-谷氨酸210 215 220赖氨酸-苏氨酸-缬氨酸-丙氨酸-脯氨酸-苏氨酸-谷氨酸-半胱氨酸-丝氨酸225 230<210> 4<211> 233<212> PRT<213> Homo sapiens<400> 4Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Pro Pro Leu SerVal Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 5 <211> 233 <212> PRT <213> Homo sapiens (Humansapiens)<400> 5Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Ser Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln ValThr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 6<211> 233<212> PRT<213> Homo sapiens<400> 6Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Gln Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser AsnAsn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 7<211> 233<212> PRT<213> Homo sapiens<400> 7Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro甘氨酸 丙氨酸 缬氨酸 苏氨酸 缬氨酸 丙氨酸 色氨酸 赖氨酸 丙氨酸 天冬氨酸 丝氨酸 丝氨酸 脯氨酸 缬氨酸 165 170 175 赖氨酸 丙氨酸 甘氨酸 缬氨酸 谷氨酸 苏氨酸 苏氨酸 苏氨酸 脯氨酸 丝氨酸 赖氨酸 谷氨酰胺 丝氨酸 天冬酰胺 天冬酰胺 赖氨酸 180 185 190 酪氨酸 丙氨酸 丙氨酸 丝氨酸 丝氨酸 酪氨酸 亮氨酸 丝氨酸 亮氨酸 苏氨酸 脯氨酸 谷氨酸 谷氨酰胺 色氨酸 赖氨酸 丝氨酸 195 200 205 组氨酸 精氨酸 丝氨酸 酪氨酸 丝氨酸 半胱氨酸 谷氨酰胺 缬氨酸 苏氨酸 组氨酸 谷氨酸 甘氨酸 丝氨酸 苏氨酸 缬氨酸 谷氨酸 210 215 220 赖氨酸 苏氨酸 缬氨酸 丙氨酸 脯氨酸 苏氨酸 谷氨酸 半胱氨酸 丝氨酸 225 230 <210> 8 <211> 233 <212> PRT <213> 智人(Homo sapiens) <400> 8 甲硫氨酸 甘氨酸 色氨酸 丝氨酸 半胱氨酸 异亮氨酸 异亮氨酸 亮氨酸 苯丙氨酸 亮氨酸 缬氨酸 丙氨酸 苏氨酸 丙氨酸 苏氨酸 甘氨酸 1 5 10 15 缬氨酸 组氨酸 丝氨酸 丝氨酸 酪氨酸 缬氨酸 精氨酸 脯氨酸 亮氨酸 丝氨酸 缬氨酸 丙氨酸 亮氨酸 甘氨酸 谷氨酸 苏氨酸 20 25 30 丙氨酸 精氨酸 异亮氨酸 丝氨酸 半胱氨酸 甘氨酸 精氨酸 谷氨酰胺 丙氨酸 亮氨酸 甘氨酸 丝氨酸 精氨酸 丙氨酸 缬氨酸 谷氨酰胺 35 40 45 色氨酸 酪氨酸 谷氨酰胺 组氨酸 精氨酸 脯氨酸 甘氨酸 谷氨酰胺 丙氨酸 脯氨酸 异亮氨酸 亮氨酸 亮氨酸 异亮氨酸 酪氨酸 天冬酰胺 50 55 60 天冬酰胺 谷氨酰胺 天冬氨酸 精氨酸 脯氨酸 丝氨酸 甘氨酸 异亮氨酸 脯氨酸 谷氨酸 精氨酸 苯丙氨酸 丝氨酸 甘氨酸 苏氨酸 天冬酰胺 65 70 75 80 天冬氨酸 异亮氨酸 天冬酰胺 苯丙氨酸 甘氨酸 苏氨酸 精氨酸 丙氨酸 苏氨酸 亮氨酸 苏氨酸 异亮氨酸 丝氨酸 甘氨酸 缬氨酸 谷氨酸 85 90 95 丙氨酸 甘氨酸 天冬氨酸 谷氨酸 丙氨酸 天冬氨酸 酪氨酸 酪氨酸 半胱氨酸 组氨酸 甲硫氨酸 色氨酸 天冬氨酸 丝氨酸 精氨酸 丝氨酸 100 105 110 甘氨酸 苯丙氨酸 丝氨酸 色氨酸 丝氨酸 苯丙氨酸 甘氨酸 甘氨酸 丙氨酸 苏氨酸 精氨酸 亮氨酸 苏氨酸 缬氨酸 亮氨酸 甘氨酸 115 120 125 谷氨酰胺 脯氨酸 赖氨酸 丙氨酸 丙氨酸 脯氨酸 丝氨酸 缬氨酸 苏氨酸Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 9 <211> 233 <212> PRT <213> Homo sapiens <400> 9 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ser Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser ArgSer100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 10<211> 233<212> PRT<213> Homo sapiens<400> 10Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp IleGly Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 11<211> 233<212> PRT<213> Homo sapiens<400> 11Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro GlyGln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Thr Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 12<211> 233<212> PRT<213> Homo sapiens<400> 12Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser ValAla Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Glu Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 13 <211> 233 <212> PRT <213> Homo sapiens (Humansapiens)<400> 13Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln ValThr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 14 <211> 233 <212> PRT <213> Homo sapiens <400> 14 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser AsnAsn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 15<211> 233<212> PRT<213> Homo sapiens<400> 15Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160TyrPro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 16 <211> 233 <212> PRT <213> Homo sapiens <400> 1 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser ValThr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 17<211> 233<212> PRT<213> Homo sapiens<400> 17Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp SerArg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 18<211> 233<212> PRT<213> Homo sapiens<400> 18Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80AspIle Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 19<211> 233<212> PRT<213> Homo sapiens<400> 19Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg ProGly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 20 <211> 233 <212> PRT <213> Homo sapiens <400> 20 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Tyr Val Arg Pro Leu SerVal Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 21 <211> 233 <212> PRT <213> Homo sapiens (Homosapiens)<400> 21Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln ValThr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 22<211> 233<212> PRT<213> Homo sapiens<400> 22Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser AsnAsn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 23<211> 233<212> PRT<213> Homo sapiens<400> 23Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160TyrPro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 24 <211> 233 <212> PRT <213> Homo sapiens <400> 2 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser ValThr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 25<211> 233<212> PRT<213> Homo sapiens<400> 25Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp SerArg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 26<211> 233<212> PRT<213> Homo sapiens<400> 26Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80AspIle Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 27<211> 233<212> PRT<213> Homo sapiens<400> 27Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 2D 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro It should be noted that there seems to be an incorrect "2D" in the translated sequence which might be a typo in the original input. The correct translation for the amino acid sequence part should be as above with proper amino acid names and numbering maintained.Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 28<211> 233<212> PRT<213> Homo sapiens<400> 28Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu SerVal Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 29 <211> 233 <212> PRT <213> Homo sapiens (Homosapiens)<400> 29Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln ValThr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 30<211> 233<212> PRT<213> Homo sapiens<400> 30Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser AsnAsn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 31<211> 233<212> PRT<213> Homo sapiens<400> 31Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160TyrPro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 32 <211> 233 <212> PRT <213> Homo sapiens <400> 32 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser ValThr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 33<211> 233<212> PRT<213> Homo sapiens<400> 33Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp SerArg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 34<211> 233<212> PRT<213> Homo sapiens<400> 34Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80AspIle Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 35 <211> 233 <212> PRT <213> Homo sapiens <400> 35 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg ProGly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 36 <211> 233 <212> PRT <213> 智人(Homo sapiens) <400> 36 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Pro Val Arg Pro Leu Ser 甘氨酸 谷氨酰胺 丙氨酸 脯氨酸 异亮氨酸 亮氨酸 亮氨酸 异亮氨酸 酪氨酸 天冬酰胺 50 55 60 天冬酰胺 谷氨酰胺 天冬氨酸 精氨酸 脯氨酸 丝氨酸 甘氨酸 异亮氨酸 脯氨酸 谷氨酸 精氨酸 苯丙氨酸 丝氨酸 甘氨酸 苏氨酸 脯氨酸 65 70 75 80 天冬氨酸 异亮氨酸 天冬酰胺 苯丙氨酸 甘氨酸 苏氨酸 精氨酸 丙氨酸 苏氨酸 亮氨酸 苏氨酸 异亮氨酸 丝氨酸 甘氨酸 缬氨酸 谷氨酸 85 90 95 丙氨酸 甘氨酸 天冬氨酸 谷氨酸 丙氨酸 天冬氨酸 酪氨酸 酪氨酸 半胱氨酸 组氨酸 甲硫氨酸 色氨酸 天冬氨酸 丝氨酸 精氨酸 丝氨酸 100 105 110 甘氨酸 苯丙氨酸 丝氨酸 色氨酸 丝氨酸 苯丙氨酸 甘氨酸 甘氨酸 丙氨酸 苏氨酸 精氨酸 亮氨酸 苏氨酸 缬氨酸 亮氨酸 甘氨酸 115 120 125 谷氨酰胺 脯氨酸 赖氨酸 丙氨酸 丙氨酸 脯氨酸 丝氨酸 缬氨酸 苏氨酸 亮氨酸 苯丙氨酸 脯氨酸 脯氨酸 丝氨酸 丝氨酸 谷氨酸 130 135 140 谷氨酸 亮氨酸 谷氨酰胺 丙氨酸 天冬酰胺 赖氨酸 丙氨酸 苏氨酸 亮氨酸 缬氨酸 半胱氨酸 亮氨酸 异亮氨酸 丝氨酸 天冬氨酸 苯丙氨酸 145 150 155 160 酪氨酸 脯氨酸 甘氨酸 丙氨酸 缬氨酸 苏氨酸 缬氨酸 丙氨酸 色氨酸 赖氨酸 丙氨酸 天冬氨酸 丝氨酸 丝氨酸 脯氨酸 缬氨酸 165 170 175 赖氨酸 丙氨酸 甘氨酸 缬氨酸 谷氨酸 苏氨酸 苏氨酸 苏氨酸 脯氨酸 丝氨酸 赖氨酸 谷氨酰胺 丝氨酸 天冬酰胺 天冬酰胺 赖氨酸 180 185 190 酪氨酸 丙氨酸 丙氨酸 丝氨酸 丝氨酸 酪氨酸 亮氨酸 丝氨酸 亮氨酸 苏氨酸 脯氨酸 谷氨酸 谷氨酰胺 色氨酸 赖氨酸 丝氨酸 195 200 205 组氨酸 精氨酸 丝氨酸 酪氨酸 丝氨酸 半胱氨酸 谷氨酰胺 缬氨酸 苏氨酸 组氨酸 谷氨酸 甘氨酸 丝氨酸 苏氨酸 缬氨酸 谷氨酸 210 215 220 赖氨酸 苏氨酸 缬氨酸 丙氨酸 脯氨酸 苏氨酸 谷氨酸 半胱氨酸 丝氨酸 225 230 <210> 36 <211> 233 <212> PRT <213> Homo sapiens <400> 36 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Pro Val Arg Pro Leu SerVal Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 37 <211> 233 <212> PRT <213> Homo sapienssapiens)<400> 37Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln ValThr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 38<211> 233<212> PRT<213> Homo sapiens<400> 38Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser AsnAsn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 39<211> 233<212> PRT<213> Homo sapiens<400> 39Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160TyrPro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 40 <211> 233 <212> PRT <213> Homo sapiens <400> 40 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val苏氨酸 亮氨酸 苯丙氨酸 脯氨酸 脯氨酸 丝氨酸 丝氨酸 谷氨酸130 135 140谷氨酸 亮氨酸 谷氨酰胺 丙氨酸 天冬酰胺 赖氨酸 丙氨酸 苏氨酸 亮氨酸 缬氨酸 半胱氨酸 亮氨酸 异亮氨酸 丝氨酸 天冬氨酸 苯丙氨酸145 150 155 160酪氨酸 脯氨酸 甘氨酸 丙氨酸 缬氨酸 苏氨酸 缬氨酸 丙氨酸 色氨酸 赖氨酸 丙氨酸 天冬氨酸 丝氨酸 丝氨酸 脯氨酸 缬氨酸165 170 175赖氨酸 丙氨酸 甘氨酸 缬氨酸 谷氨酸 苏氨酸 苏氨酸 苏氨酸 脯氨酸 丝氨酸 赖氨酸 谷氨酰胺 丝氨酸 天冬酰胺 天冬酰胺 赖氨酸180 185 190酪氨酸 丙氨酸 丙氨酸 丝氨酸 丝氨酸 酪氨酸 亮氨酸 丝氨酸 亮氨酸 苏氨酸 脯氨酸 谷氨酸 谷氨酰胺 色氨酸 赖氨酸 丝氨酸195 200 205组氨酸 精氨酸 丝氨酸 酪氨酸 丝氨酸 半胱氨酸 谷氨酰胺 缬氨酸 苏氨酸 组氨酸 谷氨酸 甘氨酸 丝氨酸 苏氨酸 缬氨酸 谷氨酸210 215 220赖氨酸 苏氨酸 缬氨酸 丙氨酸 脯氨酸 苏氨酸 谷氨酸 半胱氨酸 丝氨酸225 230<210> 41<211> 233<212> PRT<213> 智人(Homo sapiens)<400> 41甲硫氨酸 甘氨酸 色氨酸 丝氨酸 半胱氨酸 异亮氨酸 异亮氨酸 亮氨酸 苯丙氨酸 亮氨酸 缬氨酸 丙氨酸 苏氨酸 丙氨酸 苏氨酸 甘氨酸1 5 10 15缬氨酸 组氨酸 丝氨酸 丝氨酸 酪氨酸 缬氨酸 精氨酸 脯氨酸 亮氨酸 丝氨酸 缬氨酸 丙氨酸 亮氨酸 甘氨酸 谷氨酸 苏氨酸20 25 30丙氨酸 精氨酸 异亮氨酸 丝氨酸 半胱氨酸 甘氨酸 精氨酸 谷氨酰胺 丙氨酸 亮氨酸 甘氨酸 丝氨酸 精氨酸 丙氨酸 缬氨酸 谷氨酰胺35 40 45色氨酸 酪氨酸 谷氨酰胺 组氨酸 精氨酸 脯氨酸 甘氨酸 谷氨酰胺 丙氨酸 脯氨酸 异亮氨酸 亮氨酸 亮氨酸 异亮氨酸 酪氨酸 天冬酰胺50 55 60天冬酰胺 谷氨酰胺 天冬氨酸 精氨酸 脯氨酸 丝氨酸 甘氨酸 异亮氨酸 脯氨酸 谷氨酸 精氨酸 苯丙氨酸 丝氨酸 甘氨酸 苏氨酸 脯氨酸65 70 75 80天冬氨酸 异亮氨酸 天冬酰胺 苯丙氨酸 甘氨酸 苏氨酸 精氨酸 丙氨酸 苏氨酸 亮氨酸 苏氨酸 异亮氨酸 丝氨酸 甘氨酸 缬氨酸 谷氨酸85 90 95丙氨酸 甘氨酸 天冬氨酸 谷氨酸 丙氨酸 天冬氨酸 酪氨酸 酪氨酸 半胱氨酸 组氨酸 甲硫氨酸 色氨酸 天冬氨酸 丝氨酸Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 42<211> 233<212> PRT<213> Homo sapiens<400> 42Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80AspIle Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 43 <211> 233 <212> PRT <213> Homo sapiens <400> 43 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg ProGly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 44 <211> 233 <212> PRT <213> Homo sapiens <400> 44 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Pro Val Arg Pro Leu SerVal Ala Leu Gly Glu Thr 20 25 30 Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln 35 40 45 Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn 50 55 60 Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro 65 70 75 80 Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu 85 90 95 Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser 100 105 110 Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 45 <211> 233 <212> PRT <213> Homo sapiens (Homosapiens)<400> 45Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln ValThr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 46<211> 233<212> PRT<213> Homo sapiens<400> 46Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val165 170 175Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser AsnAsn Lys180 185 190Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser195 200 205His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu210 215 220Lys Thr Val Ala Pro Thr Glu Cys Ser225 230<210> 47<211> 233<212> PRT<213> Homo sapiens<400> 47Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ser Pro Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr20 25 30Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln35 40 45Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn50 55 60Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro65 70 75 80Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu85 90 95Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser100 105 110Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Gly115 120 125Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu130 135 140Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 48 <211> 481 <212> PRT <213> Homo sapiens <400> 48 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr TyrSer Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu450 455 460Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 49<211> 481<212> PRT<213> Homo sapiens<400> 49Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp ArgGlu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 50<211> 481<212> PRT<213> Homo sapiens<400> 50Met Gly Trp Ser Cys Ile Ile LeuPhe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220SerSer Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe PheLeu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 51 <211> 481 <212> PRT <213> Homo sapiens <400> 51 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 145 150 155 160 Pro Leu AlaPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro ArgGlu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 52<211> 481<212> PRT<213> Homo sapiens<400> 52Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu AsnSer Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val GluVal His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 53<211> 481<212> PRT<213> Homo sapiens<400> 53Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu SerVal Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys AspLys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His SerHis Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys<210> 54<211> 481<212> PRT<213> Homo sapiens <400> 54 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 145 150 155 160 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 165 170 175 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 180 185190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly PheTyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 55<211> 481<212> PRT<213> Homo sapiens<400> 55Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly GluPhe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn Gly Lys Glu 340 345 350 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 355 360 365 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 370 375 380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 385 390 395 400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 405 410 415 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420 425 430 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 56 <211> 481 <212> PRT <213> Homo sapiens <400> 56 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile GlyTyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp ThrLeu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 57<211> 481<212> PRT<213> Homo sapiens<400> 57Met Gly Trp SerCys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 58 <211> 481 <212> PRT <213> Homo sapiens <400> 58 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 145 150 155160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr 370 375 380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 385 390 395 400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 405 410 415 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420 425 430 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 59 <211> 481 <212> PRT <213> Homo sapiens <400> 59 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser LeuLys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 60<211> 481<212> PRT<213> Homo sapiens<400> 60Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser GluThr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerCys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala LeuHis Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys<210> 61<211> 481<212> PRT<213> Homo sapiens <400> 61 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Gly Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 145 150 155 160 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 165 170 175 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 180185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 62<211> 481<212> PRT<213> Homo sapiens<400> 62Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe GlyGlu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu 340 345 350 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 355 360 365 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 370 375 380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 385 390 395 400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 405 410 415 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420 425 430 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 63 <211> 481 <212> PRT <213> Homo sapiens <400> 63 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp IleGly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AspThr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 64<211> 481<212> PRT<213> Homo sapiens<400> 64Met Gly TrpSer Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Val Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 65 <211> 481 <212> PRT <213> Homo sapiens <400> 65 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 145 150 155160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr 370 375 380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 385 390 395 400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 405 410 415 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420 425 430 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 66 <211> 481 <212> PRT <213> Homo sapiens <400> 66 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser LeuLys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Arg Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 67<211> 481<212> PRT<213> Homo sapiens<400> 67Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser GluThr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Gln Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerCys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala LeuHis Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 68<211> 481<212> PRT<213> Homo sapiens<400> 68Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Gln Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 69<211> 481<212> PRT<213> Homo sapiens<400> 69Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Val Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Arg Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe GlyGlu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu 340 345 350 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 355 360 365 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 370 375 380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 385 390 395 400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 405 410 415 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420 425 430 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 70 <211> 481 <212> PRT <213> Homo sapiens <400> 70 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp IleGly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Phe Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Arg Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AspThr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 71<211> 481<212> PRT<213> Homo sapiens<400> 71Met Gly TrpSer Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Phe Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Arg Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 72 <211> 481 <212> PRT <213> Homo sapiens <400> 72 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 145 150 155160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr 370 375 380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 385 390 395 400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 405 410 415 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420 425 430 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 73 <211> 481 <212> PRT <213> Homo sapiens <400> 73 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser LeuLys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 74<211> 481<212> PRT<213> Homo sapiens<400> 74Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser GluThr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Phe Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerCys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala LeuHis Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys<210> 75<211> 481<212> PRT<213> Homo sapiens <400> 75 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln 85 90 95 Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Arg Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 145 150 155 160 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 165 170 175 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 180185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 76<211> 481<212> PRT<213> Homo sapiens<400> 76Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe GlyGlu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu 340 345 350 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 355 360 365 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 370 375 380 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 385 390 395 400 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 405 410 415 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 420 425 430 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 77 <211> 481 <212> PRT <213> Homo sapiens <400> 77 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp IleGly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Phe Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AspThr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu450 455 460Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys<210> 78<211> 481<212> PRT<213> Homo sapiens<400> 78Met Gly TrpSer Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys20 25 30Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met35 40 45Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu50 55 60Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro65 70 75 80Ser Leu Asn Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln85 90 95Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr100 105 110Tyr Cys Ala Arg Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser115 120 125Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly130 135 140Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe145 150 155 160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 435 440 445 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 450 455 460 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 480 Lys <210> 79 <211> 481 <212> PRT <213> Homo sapiens <400> 79 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met 35 40 45 Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro 65 70 75 80 Ser Leu Asn Ser Arg Val Val Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser 115 120 125 Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 145 150 155160Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu165 170 175Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp180 185 190Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu195 200 205Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser210 215 220Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro225 230 235 240Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met I...

Claims

1. An isolated anti-HIV antibody or antigen-binding portion thereof, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region consists of SPVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPILLIYNNQDRP SGIPERFSGTPDINFGTRATLTISGVEAGDEADYYCHMWDSRSGFSWSFG GATRLTVLG and the heavy chain variable region consists of QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKGLEWIG YISDRESATYNPSLNSRVVISVDTSKNQLSLKLNSVTPADTAVYYCARAR RGQRIYGVVSFGEFFYYYSMDVWGKGTTVTVSS; wherein the light chain variable region consists of SYVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPILLIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEADYYCHMWDSRSGFSWSFGGATRLTVLG, and the heavy chain variable region consists of QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKGLEWIG YISDRESATYNPSLNSRVTISRDTSKNQFSLKLNSVTPADTAVYYCARARR GQRIYGVVSFGEFFYYYSMDVWGKGTTVTVSS; or wherein the light chain variable region consists of SPVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPILLIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEADYYCHMWDSRSGFSWSFGGATRLTVLG, and the heavy chain variable region consists of QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKGLEWIG YISDRESATYNPSLNSRVTISRDTSKNQFSLKLNSVTPADTAVYYCARARR GQRIYGVVSFGEFFYYYSMDVWGKGTTVTVSS.

2. The isolated anti-HIV antibody, or antigen-binding portion thereof, of claim 1, wherein the light chain variable region comprises a LmdV:Y2P substitution, and the heavy chain variable region comprises a HV:R82V substitution and a HV:T108R substitution.

3. The isolated anti-HIV antibody or antigen-binding portion thereof of claim 1, wherein the heavy chain variable region comprises an HV:V79T substitution, an HV:L89F substitution, and an HV:T108R substitution.

4. The isolated anti-HIV antibody, or antigen-binding portion thereof, of claim 1, wherein the light chain variable region comprises a LmdV:Y2P substitution, and the heavy chain variable region comprises a HV:V79T substitution, a HV:L89F substitution, and a HV:T108R substitution.

5. The isolated anti-HIV antibody or antigen-binding portion thereof according to any one of claims 1 to 4, comprising: a light chain consisting of SEQ ID NO: 22, and a heavy chain consisting of SEQ ID NO: 69; a light chain consisting of SEQ ID NO: 23, and a heavy chain consisting of SEQ ID NO: 70; or The light chain consists of SEQ ID NO:24, and the heavy chain consists of SEQ ID NO:

71.

6. A pharmaceutical composition comprising the isolated anti-HIV antibody or antigen-binding portion thereof according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition further comprises a second therapeutic agent.

8. A nucleic acid molecule encoding the isolated anti-HIV antibody or antigen-binding portion thereof according to any one of claims 1 to 5.

9. The nucleic acid molecule of claim 8, wherein the nucleic acid molecule is a codon-optimized nucleic acid molecule.

10. A vector comprising at least one nucleic acid molecule according to claim 8 or 9.

11. A vector system comprising the vector according to claim 10.

12. A cell comprising the nucleic acid molecule of claim 8 or 9.

13. A method for preparing a recombinant anti-HIV antibody or an antigen-binding portion thereof, comprising: a. Obtaining the cell according to claim 12; b. culturing the cells in a culture medium under conditions that allow expression of the polypeptide encoded by the vector and assembly of the antibody or fragment thereof; and c. Purifying the antibody or fragment from cultured cells or cell culture medium.

14. Use of a first therapeutic agent comprising a therapeutically effective amount of at least one anti-HIV antibody or antigen-binding portion thereof according to any one of claims 1 to 5 for the preparation of a medicament for preventing or treating HIV infection or HIV-related diseases in a patient in need thereof.

15. The use of claim 14, wherein the medicament further comprises a second therapeutic agent.

16. The use of claim 15, wherein the second therapeutic agent is administered before, simultaneously with, or after administration of the anti-HIV antibody or antigen-binding portion thereof.

17. The use of claim 15 or 16 or the pharmaceutical composition of claim 7, wherein the second therapeutic agent is an anti-HIV-1 broadly neutralizing antibody (bNAb).

18. The use according to claim 17, wherein the anti-HIV-1 broadly neutralizing antibody is 3BNC117.

19. A kit comprising a pharmaceutically effective amount of at least one pharmaceutically acceptable dosage unit of the isolated anti-HIV antibody or antigen-binding portion thereof according to any one of claims 1 to 5.

20. The kit of claim 19, further comprising a pharmaceutically acceptable dosage unit of a pharmaceutically effective amount of an anti-HIV agent, wherein the two pharmaceutically acceptable dosage units may optionally be in the form of a single pharmaceutically acceptable dosage unit.

21. The kit of claim 20, wherein the anti-HIV agent is selected from the group consisting of non-nucleoside reverse transcriptase inhibitors, protease inhibitors, entry or fusion inhibitors, and integrase inhibitors.

22. The kit of claim 20, wherein the anti-HIV agent is an anti-HIV broadly neutralizing antibody.

23. The kit of claim 22, wherein the anti-HIV broadly neutralizing antibody is 3BNC117.

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