CN114478781A

CN114478781A - Anti-coagulation factor XI antibodies

Info

Publication number: CN114478781A
Application number: CN202111466510.4A
Authority: CN
Inventors: 王文义; 于泉; 刘小五; 徐立忠; 杜治强
Original assignee: Shanghai Benemae Pharmaceutical Corp
Current assignee: Shanghai Benemae Pharmaceutical Corp
Priority date: 2018-08-09
Filing date: 2018-08-09
Publication date: 2022-05-13
Anticipated expiration: 2038-08-09
Also published as: CN114478782A; WO2020029179A1; JP2021534098A; CA3108708A1; BR112021002472A2; MX2021001613A; CN114478781B; CN114478782B; CN113227150A; EP3833692A1; AU2018436195A1; CN116554334A; KR20210042352A; CN113227150B

Abstract

The present technology discloses antibodies that bind to Factor Xi (FXI) and/or its activated form factor xia (FXIa), or fragments of FXI and/or FXIa, and compositions containing the same. Also disclosed are methods of making the antibodies, and uses of the antibodies for making medicaments for treating and/or preventing coagulation-related disorders (e.g., thrombosis and thrombosis-related complications or disorders).

Description

Anti-coagulation factor XI antibodies

The application is a divisional application of an invention patent application with the application number of 201880098606.X and the name of 'anticoagulant factor XI antibody' in China national stage, which is submitted on 8,9 and 8 in 2018.

Technical Field

The present invention relates to antibodies capable of binding to Factor Xi (FXI) and/or its activated factor xia (FXIa) and FXI and/or FXIa fragments and uses thereof, including the use as anticoagulant agents for the treatment of thrombosis without affecting the hemostatic function.

Background

Thrombosis is a condition in which, after coagulation of blood in a blood vessel, the passage of blood flow in the affected area is blocked or impeded. If a blood clot travels along the circulatory system to a critical part of the body, serious complications may result, such as progression to the heart, brain and lungs, which may cause heart attack, stroke and pulmonary embolism. Thrombosis is a major cause of most strokes and myocardial infarctions, Deep Vein Thrombosis (DVT), pulmonary embolism, and other cardiovascular events.^1,2Thrombosis may be treated or prevented by anticoagulants (e.g., low molecular weight heparin, warfarin, and direct factor Xa inhibitors). The most common adverse effect of these currently available therapies is impaired hemostatic function.^3-5These treatment methods are limited by dose and patient compliance due to the need for close monitoring of the subject after treatment.

Therefore, there is a need for an effective preventive or therapeutic agent for thrombosis with minimal side effects. The present invention satisfies this need in the art.

Disclosure of Invention

Certain embodiments of the invention provide antibodies that bind to Factor Xi (FXI) and/or its activated form factor xia (FXIa), and FXI and/or FXIa fragments. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a recombinant antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is an immunologically active portion of an immunoglobulin molecule, such as a Fab, Fvs, or scFv. In certain embodiments, the antibody binds to the a3 domain of FXI and/or FXIa. In some embodiments, the antibody comprises one or more CDR sequences comprising or consisting of SEQ ID NOs:11-16,27-32,43-48,59-64,75-80,91-96,107-112,123-128,139-144,155-160, 171-176 and 187-192.

The present invention provides pharmaceutical compositions for the treatment and/or prevention of thrombosis and/or complications or diseases associated with thrombosis. The pharmaceutical composition comprises one or more anti-FXI and/or anti-FXIa antibodies disclosed herein. In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable adjuvants, carriers, excipients, preservatives, or combinations thereof.

The invention provides nucleic acids encoding the anti-FXI and/or anti-FXIa antibodies disclosed herein, or any antibody functional fragment, as well as vectors comprising the nucleic acids, and host cells comprising the vectors. In certain embodiments, the vector is an expression vector capable of producing an antibody or functional fragment thereof encoded by the nucleic acid in a host cell.

The present invention provides a kit comprising one or more anti-FXI and/or anti-FXIa antibodies disclosed herein for use in the treatment and/or prevention of thrombosis and/or complications or disorders associated with thrombosis. Alternatively, the kit comprises a pharmaceutical composition comprising one or more anti-FXI and/or anti-FXIa antibodies disclosed in the specification for the treatment and/or prevention of a thrombus and/or a complication or disorder associated with a thrombus. In certain embodiments, the kit further comprises instructions for use.

The present invention provides methods for treating and/or preventing thrombosis and/or complications or disorders associated with thrombosis. The methods comprise administering to a subject in need thereof an effective dose of one or more anti-FXI and/or anti-FXIa antibodies disclosed herein. Alternatively, the method comprises administering to a subject in need thereof an effective dose of a pharmaceutical composition comprising an anti-FXI antibody, an anti-FXIa antibody or a functional fragment of either antibody.

There is provided the use of an anti-FXI and/or anti-FXIa antibody as disclosed in the specification for the manufacture of a medicament for the treatment and/or prevention of thrombosis and/or a complication or disorder associated with thrombosis.

The present invention provides a method for the preparation of anti-FXI and/or anti-FXIa antibodies as disclosed in the present specification. The method takes steps that include transfecting a host cell with a vector encoding a nucleotide for the antibody and expressing the antibody in the host cell. The method may further comprise purifying the expressed antibody from the host cell. In addition, the purified antibodies can be modified such that the modified recombinant antibodies retain the activity of the corresponding human antibodies. Alternatively, the antibodies disclosed herein can be produced from hybridoma cultures.

Drawings

FIGS. 1A-1E show the anticoagulation effect of five anti-FXI antibodies in human plasma as determined by the APTT assay. 5 different antibodies were added to human plasma at 0 to 400nM for APTT detection as described in example 3. The five antibodies tested included 19F6(a), 34F8(B), 42a5(C), 1a6(D), and 14E11 (E). In this experiment, antibodies 1a6 and 14E11 were used as positive controls.

FIGS. 2A-2C show the anticoagulant effect of antibodies 19F6(A), 34F8(B) and 42A5(C) as measured by APTT in monkey plasma. 3 different antibodies were added to monkey plasma at 0 to 400nM for APTT detection as described in example 4.

FIGS. 3A-3F show SPR sensorgrams of FXI binding to immobilized h-19F6(A), h-34F8(B), and h-42A5(C), and SPR sensorgrams of FXIa binding to immobilized h-19F6(D), h-34F8(E), and h-42A5 (F). The data fit to the 1:1 binding model, and curve fitting was performed at the tested concentration range of FXI (0.005-1 ng/mL). Each curve represents a different test concentration of FXI or FXIa.

FIGS. 4A-4C show concentration-response curves for the antibodies h-19F6(A), h-34F8(B), and h-42A5(C) inhibiting hydrolysis of S-2366 by human FXIa.

FIGS. 5A-5B show the inhibitory effect of antibodies h-19F6(A) and h-42A5(B) on the FXIa-mediated FIX activation to FIXa reaction. Human FIX (200nM) and FXIa (5nM) and 1. mu. M h-19F6 or h-42A5 were placed in a medium containing 5mM CaCl₂In PBS (5), incubated at room temperature. Samples were collected at designated time intervals, and the amount of FIX and FIXa proteins in the samples were determined by western blotting using goat anti-human FIX IgG (Affinity Biologicals). FIGS. 5C-5D illustrate the inhibitory effect of antibodies h-19F6(C) and h-42A5(D) on FXIIa-mediated activation of FXI to FXIa. Human FXI (500nM) was incubated with FXIIa (50nM) in solutions containing 1. mu. M h-19F6 or h-42A 5. The amount of FXI and protein representing the FXIa light chain produced by FXIa was detected at different time points by western blot method. Human IgG4 (1. mu.M) was used as a control.

FIGS. 6A-6C show the anticoagulant effect of the antibodies h-34F8, h-19F6 and h-42A5 on APTT in cynomolgus monkeys. Monkeys were administered intravenously at the indicated doses of h-34F8(A), h-19F6(B), and h-42A5 (C). APTT clotting times of ex vivo plasma were determined before dosing (time 0) and at 0.5, 1, 3, 6, 12 and 24 hours post-dosing.

FIGS. 7A-7C show the effect of antibodies h-34F8, h-19F6, and h-42A5 on cynomolgus monkey PT. Monkeys were administered intravenously at the indicated doses of h-34F8(A), h-19F6(B), and h-42A5 (C). The PT clotting time T in ex vivo plasma was determined before dosing (time 0) and at 0.5, 1, 3, 6, 12 and 24 hours after dosing.

FIGS. 8A-8C show the effect of antibodies h-34F8, h-19F6, and h-42A5 on cynomolgus monkey Arteriovenous (AV) shunt thrombosis formation. Monkey were administered intravenously with increasing doses of h-34F8(A), h-19F6(B) or h-42A5(C) (n-3 for h-34F8 and h-19F6 and n-4 for h-42A 5) and pre-and post-dose weight changes were determined for the AV shunt thromboplastin monkey model. P <0.05, P <0.01 and P <0.001, vs. vehicle control group.

FIGS. 9A-9C show the effect of antibodies h-34F8, h-19F6, and h-42A5 on cynomolgus monkey bleeding time. Monkeys were administered intravenously with increasing doses of h-34F8(a), h-19F6(B), or h-42a5(C) (n-3 for the 34F8 and h-19F6 groups and n-4 for the h-42a5 group), and bleeding times were calculated pre-dose and 30 minutes after each dose.

FIGS. 10A-10B show the antithrombotic effects of antibodies h-34F8, h-19F6, and h-42A 5. Four groups of monkeys (n-5) were administered vehicle control, 0.3mg/kg of h-34F8, h-19F6, or h-42A5 intravenously with FeCl 2 hours after administration₃Administration ofThe left femoral artery of each animal was treated to induce thrombosis. The time to reach 80% thrombotic occlusion (a) and 100% thrombotic occlusion (B) was determined by monitoring blood flow rate. P<0.05 and P<Vehicle control group 0.01vs.

FIGS. 11A-11D show that treatment with antibodies h-34F8, h-19F6, or h-42A5 did not prolong bleeding time in monkeys. Four groups of monkeys (n ═ 5) were administered intravenously with vehicle control, 0.3mg/kg of h-34F8, h-19F6, or h-42a5, and bleeding time was measured before and 1 hour after dosing. The bleeding time of the h-34F8, h-19F6 and h-42A5 treatment groups are shown in the graphs (A), (B) and (C), respectively. The change in bleeding time between vehicle control, h-34F8, h-19F6, or h-42A5 treated groups is shown in FIG. D.

FIGS. 12A-12B show the effect of antibodies h-34F8, h-19F6, and h-42A5 on monkey plasma clotting time. Four groups of monkeys (n ═ 5) were administered vehicle control, 0.3mg/kg of h-34F8, h-19F6, and h-42a5 intravenously, respectively, and blood was collected before and about 3 hours after dosing for plasma preparation and measurement of clotting times APTT and PT. APTT changes and PT changes are shown in FIGS. (A) and (B). P <0.01 and P <0.001vs. vehicle control.

FIG. 13 is the amino acid sequence of human FXI (SEQ ID NO: 203).

FIG. 14 shows the effect of modified antibodies h-19F6 (top panel) and h-42A5 (bottom panel) on cynomolgus monkey APTT. Monkeys were given intravenous doses of modified h-19F6 and h-42A 5. APTT clotting times of ex vivo plasma were determined before dosing (time 0) and at 0.5, 2, 6, 12, 24, 48, 96, 168, 240 and 336 hours post-dose, respectively.

FIG. 15 shows the effect of modified antibodies h-19F6 (top panel) and h-42A5 (bottom panel) on cynomolgus monkey PT. Monkeys were given intravenous doses of modified h-19F6 and h-42A 5. Ex vivo plasma PT clotting time measurements were performed before (time 0) and 0.5, 2, 6, 12, 24, 48, 96, 168, 240 and 336 hours after dosing, respectively.

FIGS. 16A-16B show the effect of h-19F6 and h-42A5 on APTT and PT in human plasma. FIG. 16A shows the effect of h-19F6 and h-42A5 on APTT in human plasma. FIG. 16B shows the effect of h-19F6 and h-42A5 on PT in human plasma.

Figure 17 shows the binding specificity of the test antibody to human FXI. In western blot experiments, 10 μ L of human standard plasma, FXI-depleted human plasma were used as FXI positive and FXI negative controls, respectively.

FIG. 18 shows the effect of h-19F6 and h-42A5 on the bleeding times recorded before and 1 hour after dosing in an AV shunt thrombosis model.

FIGS. 19A-19D show the binding properties of h-19F6 and h-42A5 to human FXI. FIG. 19A shows a sensorgram of h-19F6 in mobile phase capturing a specific concentration of FXI on a sensor chip. FIG. 19B shows a sensorgram of h-42A5 in mobile phase capturing a specific concentration of FXI on the sensor chip. FIG. 19C shows the captured antibody when the tested antibody (5. mu.g/mL) was flowed over a sensor chip immobilized with equal amounts of 4 mutant FXIs, the 4 mutations being the replacement of the FXI A1, A2, A3 or A4 domain by the corresponding domain of prekallikrein, respectively. Experiment a known anti-FXI antibody O1a6 was used as a positive control. FIG. 19D shows FXI immobilized on the sensor chip. h-19F6 and h-42A5 (5. mu.g/ml) were injected sequentially into the flow cell at 30. mu.l/min and the response was monitored. Experiments were performed twice and representative results are described.

FIGS. 20A-20B show the binding properties of h-19F6 and h-42A5 to human FXIa. FIG. 20A shows a sensorgram of h-19F6 in mobile phase capturing a specific concentration of FXI on the sensor chip. FIG. 20B shows a sensorgram of h-42A5 in mobile phase capturing specific concentrations of FXI on the sensor chip.

Detailed Description

The following description of the present specification is intended only to illustrate various examples of the present invention. Therefore, the particular modifications discussed should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various equivalent substitutions, changes, and modifications can be made to the entities of the invention without departing from the scope of the invention, and it is understood that the invention also includes such equivalent examples.

The intrinsic coagulation cascade mechanism includes the intrinsic pathway and the extrinsic pathway. The intrinsic pathway, also known as the contact activation pathway,initiated by surface contact and resulting in activation of FXII. The intrinsic pathway involves FXI, FIX and FVIII. The extrinsic pathway, also known as the Tissue Factor (TF) pathway, is initiated by vascular injury and results in the formation of the TF-FVIIa activation complex. These two pathways can intersect and activate a common pathway, leading to conversion of prothrombin to thrombin, and ultimately the formation of a cross-linked fibrin clot. The ideal anticoagulant should be effective in preventing thrombosis without compromising hemostatic action. Evidence suggests that the intrinsic coagulation pathway is important for the expansion phase of coagulation, while the extrinsic and common pathways are more involved in the initiation and propagation phases.^5-8These findings indicate that the intrinsic pathway plays a secondary role in the normal hemostatic process, while inhibition of the intrinsic pathway may provide antithrombotic benefits with a lower risk of bleeding. FXI is a component of the intrinsic pathway and has recently become an attractive target as it may have the potential to elicit an antithrombotic effect without affecting bleeding.^3，5，6

FXI can be activated by factor XIIa through the intrinsic pathway to FXIa, which in turn activates factor IX. Epidemiological studies have shown that lack of FXI in humans is associated with a reduced risk of venous thromboembolism and stroke, while elevated FXI levels are associated with an increased risk of stroke.^9-11Furthermore, bleeding tendency is very low in people lacking FXI.^12，13In addition, FXI-deficient mice can be protected from multiple types of thrombosis without increasing the risk of bleeding.¹⁴Furthermore, small molecule inhibitors, antibodies and antisense oligonucleotides that inhibit FXI are antithrombotic specific and do not risk bleeding in many animal models of thrombosis.

The antibodies disclosed herein bind to FXI and/or FXIa and are directed to the intrinsic pathway of coagulation. The structure and role of FXI in coagulation has been previously reported in various documents.³³

Animal and clinical studies have shown a close association between FXI and thrombosis.³A number of groups have studied FXI deficient mice and have shown a significant antithrombotic phenotype in several models, including FeCl₃Induced arterial and deep vein thrombosis models, pulmonary embolism models, and cerebral arterial occlusion models.^{14，17，22，23}Patients with congenital FXI deficiency are not susceptible to Venous Thromboembolism (VTE) or ischemic stroke in human epidemiological studies, and patients with higher FXI levels are at higher risk of developing VTE and ischemic stroke than patients with lower FXI levels.^9-11For physiological hemostasis, the effect of FXI appears to be dispensable. FXI deficient mice did not show excessive bleeding and their tail bleeding times were comparable to wild type animals.^23，24Furthermore, patients with severe FXI lack, while possibly showing different bleeding tendencies during surgery, do not show spontaneous bleeding.^12，13Combinations of two or more antithrombotic agents are widely used clinically. One previous study showed that aspirin caused similar bleeding tendencies in wild-type and FXI deficient mice, suggesting that targeting FXI may still be safe even in the presence of other antithrombotic treatments.¹⁴

All the above findings indicate that FXI/FXIa is a safe drug target for the treatment of thrombosis related diseases without compromising hemostatic function. Thus, a number of approaches have been applied to the target FXI/FXIa to develop therapies for the treatment of thrombosis, such as antibodies, oligonucleotides and small molecule inhibitors.⁵An antibody-type blocking agent for FXI/FXIa was prepared as described in the specification. The advantages of antibodies include rapid onset of action and low frequency of administration, and the main disadvantage of antibodies is their potential immunogenicity.²⁵At least two of the tested antibodies were humanized prior to in vivo studies. The two humanized antibodies h-19F6 and h-42A5 have high affinity for human FXI/FXIa. Interestingly, they bound different regions of the same domain of FXI (a 3). Without being bound by any theory, these antibodies may both inhibit FXIa activity but have no effect on FXI activation mediated by FXIIa or thrombin.

Various types of FXI/FXIa inhibitors prolong APTT time and show antithrombotic effects in different models. anti-FXI antibody 14E11 isolation of femoral external artery and vein from baboonAPTT in the flow increased by about 1.3-fold and decreased thrombosis.¹⁷Antisense oligonucleotides that inhibit FXI expression can reduce baboon plasma FXI levels by about 50% and reduce thrombus formation.^26，27In addition, the orally bioavailable small molecule FXIa inhibitor ONO-5450598 significantly inhibited thrombosis in the monkey thrombosis model.²⁸Furthermore, antithrombotic effects against the therapeutic target FXI/FXIa have also been demonstrated in a number of non-primate animal models, such as mouse and rabbit thrombosis models.^19，29-31A recent clinical trial showed that antisense oligonucleotides against FXI can prevent venous thrombosis in knee replacement patients.³²As shown in the examples, the antithrombotic effects of h-19F6 and h-42A5 were evaluated in two different primate thrombosis models. In the AV shunt thrombosis model, both antibodies reduced thrombosis in a dose-dependent manner. In FeCl₃In the induced thrombosis model, both antibodies extended the time of thrombosis-induced vascular occlusion. These results provide further evidence of the antithrombotic effect of FXI/FXIa inhibitors. Dose-dependent reduction of thrombosis in the AV shunt thrombosis model of h-19F6 and h-42A5 indicated that thrombosis may be inversely correlated with the degree of FXI inhibition, as indicated by prolongation of APTT. Since h-42A5 is more effective in extending APTT than h-19F6, it can also be deduced that in FeCl₃Similar results were obtained when comparing the antithrombotic effects of h-42A5 and h-19F6 in an induced thrombosis model. Thus, the more pronounced the effect of FXI/FXIa inhibitors on prolonging APTT, the more potent the inhibition of FXI/FXIa, possibly leading to better antithrombotic results.

In the development of antithrombotic agents, bleeding risk is the most relevant problem. As previously mentioned, patients lacking FXI may develop a bleeding tendency in the surgical environment. It is not clear to what extent inhibition of plasma FXI activity is still safe in terms of bleeding risk. As shown in the examples, the risk of bleeding was tested that h-19F6 and h-42A5 strongly inhibited the FXI/FXIa action in the same monkey used in the thrombosis experiments. In AV shunt thrombogenic animals, with hAn increase in the therapeutic dose of-19F 6 or h-42A5, no bleeding tendency was observed, indicating that the risk of bleeding may not be related to the degree of FXI inhibition. In FeCl₃Neither h-19F6 nor h-42A5 induced excessive bleeding in thrombosed animals. h-42A5 treatment resulted in an approximately 2-fold increase in plasma APTT, indicating over 99% inhibition of FXI. Previous studies have never assessed the risk of bleeding under such strong conditions of prolonged APTT and high FXI inhibition. The antisense oligonucleotide ISIS416856 only resulted in a 30% increase in APTT when assessing its bleeding risk. In other bleeding risk assessment studies in primates, the highly potent anti-FXI antibody, axmamb, resulted in an approximately 1-fold increase in APTT (from 30.5s to 65.6 s).²⁶Thus, the results described herein indicate that strong inhibition of FXI/FXIa does not increase the bleeding risk in primates. Therefore, FXI can be used as a drug target for thrombosis therapy.

anti-FXI or anti-FXIa antibodies

The present specification provides antibodies that bind to FXI, FXIa and/or fragments of FXI or FXIa and inhibit blood clot formation. These antibodies are able to bind FXI, FXIa and/or fragments of FXI or FXIa (e.g. fragments comprising the a3 domain) and show inhibitory effects at concentrations well below the maximum safe dose. For example, in some embodiments, antibodies at doses between 0.1mg/kg i.v. to 3mg/kg i.v. exhibit inhibitory effects on the conversion of cynomolgus monkey FXI to FXIa. In addition, the antibodies disclosed in the present specification can be used as anticoagulants with excellent safety because they cause minimal risk of bleeding compared to conventional anticoagulants (e.g., heparin).

As shown in the examples, antibodies with anti-coagulant properties were determined by preparing a number of anti-human FXI antibodies by immunizing rats with human FXI. Over a dozen such antibodies were identified, some of which were humanized for further development. Humanized rat anti-human FXI antibodies, such as the h-19F6 and h-42A5 antibodies, were characterized in vitro and in vivo. In vitro studies, humanized antibodies inhibited activation of FXI (fxia) -mediated hydrolysis of FXI, but did not inhibit FXI activation induced by factor XIIa. The binding characteristics of the antibody to FXI were determined, with dissociation constants (KD) of 22pM and 35pM for h-19F6 and h-42A5, respectively. These two antibodies bind to the FXI a3 domainAt different sites in (b). In vivo studies, two different primate thrombosis models were used to assess the antithrombotic effect and bleeding risk of humanized antibodies. In the Arteriovenous (AV) shunt thrombosis model, both antibodies dose-dependently reduced thrombosis without causing bleeding. In FeCl₃In the induced thrombosis model, both antibodies extended the time to thrombosis-mediated vascular occlusion, and neither antibody increased bleeding. Both antibodies showed antithrombotic efficacy without compromising primate hemostasis, further demonstrating that targeting FXI could be used to treat thrombosis.

In this specification, the term "comprising" with respect to a composition or method means that the composition or method includes at least the recited elements. The term "consisting essentially of means that the composition or method includes the elements described, and may further include one or more additional elements that do not materially affect the novel and basic characteristics of the composition or method. For example, a composition consisting essentially of the elements may include the elements plus one or more residues from the isolation and purification process, a pharmaceutically acceptable carrier such as phosphate buffered saline, and preservatives and the like. The term "consisting of" means that the composition or method includes only the elements recited. Examples defined by each transition term are within the scope of the present invention.

The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active part thereof that specifically binds to or has specific immunoreactivity with a specific antigen, which may for example be FXI, FXIa and a specific domain or fragment of FXI or FXIa, such as the a3 domain. In certain embodiments, the antibodies of the present methods, compositions, and kits are full-length immunoglobulin molecules comprising two heavy chains and two light chains, each heavy and light chain comprising three Complementarity Determining Regions (CDRs). The term "antibody" includes, in addition to natural antibodies, genetically engineered or otherwise modified forms of immunoglobulins, such as synthetic antibodies, intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, peptibodies, and heteroconjugate antibodies (e.g.Such as bispecific antibodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies, diabodies, triabodies, and tetrabodies). The antibodies disclosed herein can be monoclonal or polyclonal. In embodiments where the antibody is an immunologically active portion of an immunoglobulin molecule, the antibody can be Fab, Fab ', Fv, Fab ' F (ab ')₂A disulfide-linked Fv, a single-chain Fv antibody (scFv), a single-domain antibody (dAb), or a diabody. Antibodies disclosed in the present specification, including as immunologically active portions of immunoglobulin molecules, retain the ability to bind to a specific antigen (e.g. FXI or FXIa) or to a specific fragment of FXI or FXIa (e.g. the a3 domain).

In some embodiments, the anti-FXI and/or anti-FXIa antibodies disclosed herein undergo post-translational modifications, such as phosphorylation, methylation, acetylation, ubiquitination, nitrosylation, glycosylation, lipidation, or the like, during protein expression in a mammalian cell line. Techniques for the production of recombinant antibodies and in vitro and in vivo modification of recombinant antibodies are known in the art. References such as mAbs 6(5):1145 and 1154(2014), the contents of which are incorporated herein by reference.

The specification also discloses polynucleotides or nucleic acids encoding the anti-FXI and/or anti-FXIa antibodies disclosed herein. In some embodiments, the polynucleotide or nucleic acid comprises DNA, mRNA, cDNA, and plasmid DNA. Nucleic acids encoding the antibodies or functional fragments thereof disclosed herein may be cloned into a vector, such as a pTT5 mammalian expression vector, which may further include a promoter and/or other transcriptional or translational control elements, such that the nucleic acids may be expressed as the antibodies or functional fragments thereof.

The present specification Discloses Nucleic Acid (DNA) and/or amino acid (PRT) sequences of some examples of antibodies, which are shown in table 1, including VH, VL and CDR sequences.

Table 1: antibody sequences

In certain embodiments of the invention there are provided humanized anti-FXI and/or anti-FXIa antibodies. Various techniques are known in the art for humanizing non-human antibodies to approximate naturally occurring antibodies in humans. There are 6 CDRs in each antigen binding domain of a natural antibody. These CDRs are short, non-contiguous amino acid sequences that are specifically positioned to form an antigen-binding domain when the antibody assumes its three-dimensional configuration. The remainder of the antigen binding domain is called the "framework" regions, which have less intermolecular variability and form scaffolds to allow the correct positioning of the CDRs. In some embodiments, the antibodies or fragments disclosed herein have a conserved sequence of CDR3 regions.

For example, humanization of the antibodies disclosed herein can be accomplished by grafting the CDRs of monoclonal antibodies produced by immunized mice or rats. The CDRs of a mouse monoclonal antibody can be grafted into a human framework, which is then linked to a human constant region to obtain a humanized antibody. Briefly, a human germline antibody sequence database, a Protein Database (PDB), an inn (international Nonproprietary names) database, and other suitable databases may be searched and the framework most closely approximating the desired antibody found by the search. In addition, some back mutations to the donor amino acid residues were made in the human acceptor framework. In some embodiments, the variable region is linked to a human IgG constant region. For example, the Fc domain of human IgG1, IgG2, IgG3, and IgG4 can be used. Based on the prior art, humanization of monoclonal antibodies produced by non-human species is a fundamental capability of one of ordinary skill in the art.

Table 2 is an example of the variable region sequences of several humanized antibodies.

Table 2: sequences of humanized antibodies

The antibodies provided herein include variants of the sequences disclosed herein that contain one or more mutations in the amino acid sequence while retaining binding affinity for FXI, FXIa, and/or fragments thereof (e.g., fragments comprising the a3 domain). In some embodiments, the variable region of the antibody comprises an amino acid fragment having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to, or retains binding affinity for FXI, FXIa and/or a fragment thereof, selected from SEQ ID NO: 9. 10, 25, 26, 41, 42, 57, 58, 73, 74, 89, 90, 105, 106, 121, 122, 137, 138, 153, 154, 169, 170, 160, 170, 185, 186 and 197, 209.

Also disclosed are nucleic acid variants comprising a nucleic acid encoding an antibody that binds FXI, FXIa, and/or a fragment thereof (e.g., a3 domain-containing fragment). In some embodiments, the nucleic acid encoding the antibody variable region comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid sequence encoding a polypeptide having binding affinity for FXI, FXIa and/or a fragment thereof selected from the group consisting of SEQ ID NO: 1.2, 17, 18, 33, 34, 49, 50, 65, 66, 81, 82, 97, 98, 113, 114, 129, 130, 145, 146, 147, 148, 161, 162, 177 and 178.

In some embodiments, the antibodies described above will be subjected to further strategic chemical, production, and control (CMC) developments to enable the novel antibodies disclosed herein (e.g., monoclonal antibodies or humanized monoclonal antibodies) to be marketed from early drug discovery, to clinical trials and subsequent drugs. The modification will further improve the properties of the antibody without compromising the immune function of the antibody. In certain embodiments, the CMC modified antibody is more stable than the unmodified antibody at a variety of temperatures (e.g., 4 ℃, 25 ℃, and 37 ℃), for longer periods of time (e.g., 3 days, 7 days, 14 days, and 28 days), and under repeated freeze/thaw cycles (e.g., -40 ℃/25 ℃ to 5 cycles). In addition, the CMC modified antibody has acceptable solubility. For example, for a given light or heavy chain sequence, certain amino acids may be potential oxidation and glycosylation sites. These amino acid residues at potential oxidation, deamidation or glycosylation sites can be mutated, and other residues in the vicinity can also be mutated and/or optimized to maintain the 3D structure and function of a particular antibody. In some embodiments, one or more amino acid residues in a CDR region with oxidative, deamidation or glycosylation potential are mutated to improve the stability of an antibody or fragment thereof without compromising immune function. In some embodiments, one or more Met residues in the CDR regions with oxidative potential are mutated. In some embodiments, one or more Asn residues in a CDR region with deamidation potential are mutated.

The sequences of the variable regions of several exemplary CMC optimized humanized antibodies are shown in table 3 below.

Table 3: sequences of CMC optimized humanized antibodies

Pharmaceutical composition

The antibodies disclosed in the present specification can be formulated into pharmaceutical compositions. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or combinations thereof. The pharmaceutical composition can be in various dosage forms, for example, injection, freeze-dried preparation, liquid preparation, etc. Depending on the dosage form and the route of administration, suitable adjuvants, carriers, excipients, preservatives and the like may be selected as additives.³⁴

The pharmaceutical composition may be included in a kit with instructions for use of the composition.

Method of treatment

The present invention provides methods for treating and/or preventing a thrombus in a subject having a thrombus and/or an elevated risk of developing a thrombus. Methods of inhibiting clot formation in a subject are also provided. These methods require administration of an effective dose of an anti-FXI and/or FXIa antibody provided by the present invention to interfere with the intrinsic pathway. In some embodiments, the methods comprise administering to the subject a pharmaceutical composition comprising an anti-FXI and/or anti-FXIa antibody provided by the present invention.

The present specification discloses methods for preventing and/or treating a thrombosis related complication or disorder in a subject in need thereof. Thrombi can lead to a number of complications or disorders, such as embolic stroke, venous thrombosis (e.g., Venous Thromboembolism (VTE), Deep Vein Thrombosis (DVT), and Pulmonary Embolism (PE)), arterial thrombosis (e.g., Acute Coronary Syndrome (ACS), Coronary Artery Disease (CAD), and Peripheral Artery Disease (PAD)). Other conditions associated with thrombosis include: for example, patients who are surgical, bedridden, cancer, heart failure, pregnant or suffering from other medical conditions that may cause thrombosis have a high risk of VTE. The methods disclosed herein relate to prophylactic anticoagulant therapy, i.e., thrombus prevention. These methods entail administering to a subject suffering from a thrombosis related complication as disclosed above an effective dose of an anti-FXI and/or FXIa antibody as disclosed herein or an effective dose of a pharmaceutical composition comprising an anti-FXI and/or FXIa antibody. The antibody or pharmaceutical composition may be administered alone or in combination with any other therapy for treating or preventing a thrombosis related complication or disorder.

The invention also provides methods of treating and/or preventing sepsis in a subject in need thereof. There have been previous attempts to administer anticoagulants to septic patients to improve mortality or morbidity. However, this attempt was not successful because the anticoagulant caused unexpected bleeding. The antibodies disclosed herein can be used as adjunctive therapy to other therapeutic agents (e.g., antibiotics) for sepsis.

The term "subject" as used herein refers to a mammalian subject, preferably a human. "subject in need thereof" refers to a subject who has been diagnosed as having, or at increased risk of developing, a thrombus or a thrombus-related complication or condition. "subject" and "patient" are used interchangeably in this specification.

The term "treating" in reference to a disorder as used herein refers to partially or completely alleviating the disorder, preventing the disorder, reducing the likelihood of the disorder developing or relapsing, slowing the progression or onset of the disorder, or eliminating, reducing or slowing the onset of one or more symptoms associated with the disorder. In the context of a thrombus and/or thrombus-related complication or condition, "treating" can refer to preventing or slowing the growth of an existing blood clot, and/or preventing or slowing the formation of a blood clot. In some embodiments, the term "treating" refers to a reduction in the number or size of blood clots in a subject as compared to a subject not administered the antibody or functional fragment thereof. In some embodiments, the term "treating" or "treatment" refers to a reduction in one or more symptoms of thrombosis and/or thrombosis related disorder or complication in a subject following treatment with an antibody or pharmaceutical composition as disclosed herein, as compared to a subject not receiving treatment.

As used herein, an "effective dose" of an antibody or pharmaceutical composition refers to the amount of the antibody or pharmaceutical composition that produces a desired therapeutic effect (e.g., treating and/or preventing thrombosis) in a subject. In certain embodiments, an effective dose is the amount of antibody or pharmaceutical composition that produces the greatest therapeutic effect. In other embodiments, the therapeutic effect of the effective dose is less than the maximum therapeutic effect. For example, an effective amount can be an amount that is both therapeutically effective and avoids one or more side effects associated with the maximum therapeutically effective amount. The effective dosage of a particular composition will vary based on a variety of factors including, but not limited to, the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (e.g., age, body weight, sex, type and stage of disease, medical history, general physical condition, response to a given dose, and other drugs currently used), the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition, and the mode of administration. Those skilled in the clinical and pharmacological arts can determine effective dosages by routine experimentation, i.e., by monitoring the subject's response to administration of the antibody or pharmaceutical composition and adjusting the dosage accordingly. For additional guidance, see, e.g., Remington: the Science and Practice of Pharmacy, 22 nd edition, Pharmaceutical Press, London, 2012, and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 12 th edition, McGraw-Hill, New York, 2011, The entire disclosure of which is incorporated herein by reference.

In some embodiments, an effective dose of an antibody disclosed herein is from about 0.01mg/kg to about 30 mg/kg, from about 0.1mg/kg to about 10mg/kg, from about 1mg/kg to about 5 mg/kg.

It is within the ability of one of ordinary skill in the art to select an appropriate route of administration, e.g., subcutaneous, intravenous, intramuscular, intradermal, intrathecal, or intraperitoneal. To provide treatment to a subject in need thereof, the antibody or pharmaceutical composition may be administered continuously or intermittently with immediate, controlled or sustained release. In addition, the antibody or pharmaceutical composition may be administered three times a day, twice a day, or once a day for a period of 3 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, or 4 weeks. The antibody or pharmaceutical composition may be administered over a predetermined period of time. Alternatively, the antibody or pharmaceutical composition may be administered until a specific therapeutic benchmark is reached. In certain embodiments, the methods provided herein comprise the step of evaluating one or more treatment benchmarks to determine whether to continue administration of the antibody or pharmaceutical composition.

Method for producing antibody

The present specification also provides methods of making the anti-FXI and/or anti-FXIa antibodies disclosed herein. In certain embodiments, these methods entail cloning a nucleic acid encoding an anti-FXI and/or anti-FXIa antibody into a vector, transforming a host cell with the vector, and culturing the host cell to express the antibody. The expressed antibody can be purified from the host cell using any known technique. A variety of expression vectors can be used, such as the pTT5 vector and the pcDNA3 vector, as well as a variety of host cell lines, such as CHO cells (e.g., CHO-K1 and ExpicHO) and HEK193T cells.

The present specification also encompasses antibodies produced by the methods disclosed above. The antibody may have undergone one or more post-translational modifications.

The following examples are provided to better illustrate the technical solution and should not be construed as limiting the scope of coverage of any claims. Reference to specific materials is for illustrative purposes only and is not intended to limit the invention. Within the scope of the present invention, equivalent means or reactants may be developed by those skilled in the art without the need for creative efforts. It should be understood that many variations of the steps described in this specification may occur while remaining within the scope of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.

Examples

Example 1: materials and methods

A material. Human FXI (Cat No. HFXI 1111), FXIa (Cat No. HFXIa 1111a), FXIIa (HFXIIa 1212a) and FIX (Cat No. HFIX 1009) were purchased from Enzyme Research Laboratory (IN, USA).

And (5) preparing an antibody. Animal immunization and hybridoma screening were performed in Genscript Inc (china, south kyo), and procedures applied to animals in this protocol were approved by the gen-instituted animal care and use committee. Experiments were performed according to approved guidelines. Wistar rats were immunized with human FXI, and spleen cells of animals with good immune response were collected to prepare hybridomas, which were then used for subcloning by limiting dilution. Finally, several monoclonal hybridoma cell clones expressing the desired anti-FXI antibodies were obtained by using ELISA and functional screening, including 19F6, h-34F8, and 42a 5. After determination of the amino acid sequences of their variable regions, 19F6, h-34F8 and 42A5 were humanized to give three humanized antibodies in the form of IgG4, h-19F6, h-34F8 and h-42A 5. These three humanized antibodies were produced in a mammalian transient expression system and purified by protein G chromatography.

Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). Standard human plasma purchased from Symens inc. was mixed with equal volumes of each antibody at concentrations from 0 to 400nM for 5 minutes, respectively, and then tested on a CA600 analyzer. In the APTT assay, 50 μ L of plasma-antibody mixture and 25 μ L of APTT reagent (SMN 10445709, Symens Inc.) were mixed for 4 minutes at 37 ℃. Then 25. mu.L of CaCl was added₂Solution (25mM, SMN 10446232, Symens Inc.) and the time to clot formation was determined. In the PT assay, 50 μ L of plasma-antibody mixture was mixed with an equal volume of PT reagent (SMN 10446442, Symens Inc.) at 37 ℃ and the time to clot formation was then determined.

The effect of the antibodies on APTT and PT in monkey plasma was also assessed using the same method as human plasma. In these assays, equal volumes of monkey plasma diluted in Phosphate Buffered Saline (PBS) were used instead of the human plasma-antibody mixture described above.

FXIIa activates FXI. Human FXI (500nM) was preincubated in PBS for 5min at room temperature with 1 μ MIgG4 control or h-19F6 or h-34F8 or h-42A 5. At time zero, FXIIa, HK and kaolin were added to give final concentrations of FXI (250nM), FXIIa (50nM), HK (100nM) and kaolin (0.5 mg/mL). Samples of 50. mu.L were collected into dodecyl sulfate sample buffer at 0, 30, 60, 120 minute intervals. The samples were size fractionated on a 10% non-reducing gel and then transferred to a polyvinylidene fluoride membrane. The FXI as well as FXIa light chain levels were determined by western blotting using mouse anti-human FXI IgG (1C5, homemade antibody binding to the FXI C-terminus). Image results were acquired using a ChemiDocMP imaging system with Image Lab software (Bio-Rad).

FXIa mediated FIX activation. Human FIX (200nM) and FXIa (5nM) in a medium containing 5mM CaCl at room temperature in an environment containing 1. mu.M IgG1 control, h-19F6, h-34F8, or h-42A5₂Was incubated in PBS (g). Every 0, 15, 30, 45 and 60 minutes, 50 μ L of sample was collected into dodecyl sulfate sample buffer. The samples were size fractionated on a 10% non-reducing gel and then transferred to a polyvinylidene fluoride membrane. Western blots were performed using goat anti-human FIX IgG (Affinity Biologicals) to determine FIX as well as FIXa levels. The Image results were acquired using a ChemiDocMP imaging system with Image Lab software (Bio-Rad).

Surface Plasmon Resonance (SPR). The interaction of the antibody with FXI was determined by SPR assay of Biacore T200 system (Biacore, GE Healthcare). Briefly, human IgG capture antibody (Biacore, GE Healthcare) was previously immobilized on a CM5 sensor chip (GE Healthcare), and the test antibody was captured by flowing through the chip. The final amount of captured test antibody was adjusted to 15 Reaction Units (RU) equally by adjusting the capture time. Then, the antigen FXI was flowed over the chip with an association time of 180 seconds and an dissociation time of 1200 seconds. FXI was tested at concentrations of 0.063, 0.313, 0.625, 1.25, 3.125, and 6.25 nM. Data were collected and the affinity between the tested antibody and FXI was analyzed using Biacore Evaluation Software.

To determine the binding site of the test antibody on FXI, four FXI mutants C-terminally labeled with 6xHis were first generated by replacing each Apple domain (a1, a2, A3, and a4) with the corresponding domain of human prekallikrein. Equal amounts of each mutant were immobilized on a CM5 sensor chip, and the antibody to be tested (33.3nM) was passed through the chip with an association time of 180s and an dissociation time of 1200 s. The amount of each antibody captured was recorded using a Biacore Evaluation Software, measured in Reaction Units (RU).

Epitope binding results of the tested antibodies were also analyzed using the Biacore T200 system. Briefly, wild-type FXI with a 6XHis tag was pre-immobilized on a CM5 sensor chip (GE Healthcare), and h-19F6, h-34F8 or h-42A5 (5. mu.g/ml) were sequentially injected into the flow cell of the sensor surface at a flow rate of 30 microliters/min to monitor the change in response.

Pharmacodynamics of cynomolgus monkey. This animal experiment and subsequent AV thrombosis experiments were performed in Wincon inc. (china, nanning), and the procedures applied to animals in this protocol were approved by the Wincon institutional animal care and use committee. Experiments were performed according to approved guidelines. Animals received varying doses of h-19F6, h-34F8 or h-42A5 as bolus injections. 2mL of blood from the upper limb superficial vein was collected into a collection tube containing 3.2% sodium citrate before dosing (time 0) and at 0.5h, 1h, 3h, 6h, 12h and 24h post-dosing. The tubes were then mixed ten times by gentle inversion at room temperature. Plasma was collected in labeled tubes and stored at-20 ℃ until clotting time analysis was complete. Plasma samples were diluted with equal volumes of phosphate buffered saline (pH 7.4) and then subjected to APTT and PT analysis on an automated analyzer (CA660, Sysmex Inc.).

AV shunt thrombosis and bleeding time test. Cynomolgus monkeys were injected intravenously with antibodies and tested 30 minutes later. Then, a tail vein bleeding time test was performed, and thrombosis was induced again. Thrombosis was induced by connecting a shunt device, containing a pre-weighed 10cm long wire, between the femoral artery and the femoral venous cannula. Blood was allowed to flow through the shunt for 10 minutes. The thrombus formed on the wire was weighed. Immediately after the shunt is removed, a blood sample is collected and given the next higher level of antibody to be tested. Four bleeding/thrombosis events were performed in the same animal, with vehicle control and three increasing dose levels of test antibody (0.1, 0.3, 1 mg/kg).

To assess bleeding time, a 2-mL syringe was inserted into the tail vein of the animal. When the amount of blood in the syringe stopped increasing, the elapsed time was manually recorded as the bleeding time.

Ferric chloride (FeCl)₃) Resulting thrombosis and bleeding time test. The animal experiment was performed in PharmaLegacy LaThe procedures for animals in this protocol were approved by the pharmacophore institute for animal care and use committee. Experiments were performed according to approved guidelines. Cynomolgus monkeys were pre-anesthetized with 1.5mg/kg Zoletil, intubated, and ventilated with a ventilator. Anesthesia was maintained with isoflurane. Blood pressure, heart rate and body temperature were monitored throughout the procedure. In the use of FeCl ₃2 hours before, the administration of either blank vehicle or 0.3mg/kg of h-19F6, h-34F8 or h-42A5 via the limb vein. The left femoral artery was exposed and isolated by blunt dissection. A doppler flow probe is mounted on the artery and the flow is continuously recorded. In the use of FeCl₃Before, the blood flow is measured for at least 5 minutes. Then, two sheets were pre-impregnated with 40% FeCl₃The filter paper of (a) was applied to the adventitial surface of the blood vessel upstream of the probe for 10 minutes. After removing the filter paper, the application site was washed with brine. Blood flow is measured continuously until blood flow is reduced to 0. The time to 80% occlusion (20% reduction in blood flow to baseline blood flow) and 100% occlusion (0 reduction in blood flow) were recorded. In the same animals, hemostatic effect was assessed using a Surgicutt device and bleeding time was manually recorded before and 1 hour after dosing. At the end of the study (approximately 3 hours post-dose), blood samples were collected.

The binding specificity of the antibody to human FXI was tested in plasma. The test antibodies (h-19F6, h-42A5 and 14E11) were first biotinylated using the EZ-LinkTM Sulfo-NHS-LC-biotinylation kit (Cat No.21435, Thermo Fisher Inc.). These antibodies (25 μ g each) were incubated with 200 μ L of human standard plasma (Siemens Inc.) or FXI-poor human plasma (Hyphen Biomed Inc.) for 1 h. Then 50. mu.L of streptavidin-coated beads (Dynabeads M-280 streptavidin, Thermo Fisher Inc.) were added to the mixture to enrich the biotin-containing antigen-antibody complexes. After 3 washes with PBS, the antigen-antibody complex was eluted and western blotted using mouse anti-human FXI IgG (1C5, home-made antibody binding to FXI C-terminus) as primary antibody. Image results were acquired using a ChemiDocMP imaging system with Image Lab software (Bio-Rad). In western blot, 10 μ L of human standard plasma and FXI-poor human plasma were used as FXI positive and FXI negative controls, respectively.

And (5) carrying out statistical analysis. Numerical data from multiple experiments are presented as mean ± Standard Error of Mean (SEM). Thrombus weight was statistically analyzed in both the AV shunt test and the two bleeding time test using one-way analysis of variance (ANOVA) followed by multiple comparative tests with Dunnett (Dunnett). Kruskal-Wallis grade test was performed to statistically analyze FeCl₃Time to occlusion in the resulting thrombosis experiment. P <0.05 is considered statistically meaningful.

Example 2: generation and sequencing of anti-FXI antibodies.

Respectively immunizing BALB/c mice and Wister rats by using human coagulation Factors XI (FXI), and taking spleen cells of animals with better immune response for preparing hybridoma cells; after the hybridoma cells are subjected to limited dilution, further subcloning is carried out, and 12 monoclonal hybridoma cell lines are successfully obtained through an ELISA capture method and functional screening, wherein the expressed anti-FXI antibodies of the monoclonal hybridoma cell lines are respectively as follows: 3G12, 5B2, 7C9, 7F1, 13F4, 19F6, 21F12, 34F8, 38E4, 42a5, 42F4, and 45H 1.

To determine the amino acid and nucleotide sequences of the light and heavy chain variable regions (VL, VH) of the above antibodies, cDNAs encoding VL and VH were cloned from the corresponding hybridoma cells according to standard PT-PCR protocols. The sequences of the corresponding antibody VL, VH (including CDR sequences) are detailed in Table 1.

Example 3: the anticoagulant activity of the antibody of the present invention in human plasma was evaluated using the Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) assay indices.

The APTT assay measures the activity of intrinsic and common pathway coagulation; whereas the PT assay measures the activity of extrinsic and common pathway coagulation. The antibodies tested in this experiment were 19F6,34F8, 42a5, 1a6 and 14E 11. In this experiment, antibodies 1a6 and 14E11 were used as positive controls. The sequences of the control antibody variable regions were derived from US patent US 8,388,959 and US patent application 2013/0171144 and reformatted as IgG 4. These antibodies were then expressed using the expihho cell system. APTT and PT assays were performed as described above.

As shown in FIG. 1, all antibodies tested increased the Activated Partial Thromboplastin Time (APTT) and exhibited some concentration dependence in a relatively low concentration range, e.g., as the concentration increased to 100nM (14E11 increased to 200 nM); but all antibodies had no significant effect on Prothrombin Time (PT) (data not provided). These results indicate that all antibodies tested inhibit the intrinsic pathway of coagulation, but not the extrinsic pathway.

Example 4: the anticoagulant activity of the antibody in non-human plasma is evaluated by using an APTT detection index.

Anticoagulant activity of different antibodies (including 19F6,34F8 and 42A5) in plasma of mice, rats and monkeys was tested as in example 3. The results show that in mouse and rat plasma all antibodies tested had no effect on APTT, but in monkey plasma their APTT values increased and were concentration-dependent over a relatively low concentration range (see figure 2 for details). The test shows that the antibody only has cross reaction on monkey FXI/FXIa and has no cross reaction on mouse FXI/FXIa and rat FXI/FXIa.

Example 5: humanization of anti-FXI antibodies

Murine monoclonal antibodies cannot be used directly for therapy due to their short half-life and induction of human anti-mouse antibody responses. One approach to solving this problem is to humanize murine antibodies. Some antibodies are humanized by CDR grafting. For each murine antibody V was determined_LAnd V_HSuitable human acceptor frameworks, and various numbers of back mutations are introduced into the selected human frameworks to maintain the structure and/or function of the resulting antibody. The humanized antibodies are considered to be successfully humanized if the affinity and function of the humanized antibodies are substantially comparable to that of the corresponding unmodified antibody. Three humanized V of 19F6,34F8, 42a5_LAnd V_HThe sequences are shown as h-19F6, h-34F8, h-42A5 and are shown in Table 2.

Example 6: affinity of anti-FXI antibodies to human FXI

The affinity of the anti-FXI antibody to FXI/FXIa was determined by Surface Plasmon Resonance (SPR) technique using BIAcore T200 system. The method comprises the following steps: the humanized antibody is constructed by connecting the variable region of the antibody to be detected with the Fc fragment of human IgG4 and is subjected to recombinant expression in CHO cells; these antibodies were bound to a Biacore CM5 sensor chip coupled to an anti-human IgG capture antibody.

Then purified antigens FXI or FXIa with different concentrations (0.005-1 mu g/ml) flow through a CM5 sensing chip, the binding time of the anti-FXI/FXIa antibody is 180s, and the dissociation time is 1800 s. The binding data collected was analyzed using Biacore Evaluation Software (supplied from GE Healthcare) to determine the affinity of FXI/FXIa and the detection antibody. The SPR maps of the immobilized h-9F6, h-34F8 and h-42A5 antibodies bound to FXI/FXIa are shown in detail in FIG. 3. As shown in FIG. 3, the Response (RU) of each antibody was also increased with the gradual increase of the concentration of FXI or FXIa. Dissociation constants (KD) were calculated for FXI and FXIa for h-19F6, h-34F8 and h-42A5 and the results are shown in Table 4. Since the difference was less than 10-fold, the affinity of each antibody for FXI and FXIa was considered to be the same.

Table 4: KD values for antibodies against FXI and FXIa

Example 7: determination of the binding site of an anti-FXI antibody on FXI

The binding site of 19F6, 42A5 on FXI was determined using SPR technique. The method comprises the following steps: the human IgG capture antibody is coupled on the surface of a Biacore CM5 sensing chip, the recombinant sample h-19F6 or h-42A5 flows through the surface of the chip to be captured, and the capture quantity of h-19F6 or h-42A5 reaches 15 relative units by adjusting the flow time. Flowing wild type FXI or chimeric FXI (i.e. FXI/PK chimera with a functional domain of FXI replaced by a corresponding domain of human prekallikrein) at a certain concentration on the surface of the chip, wherein the binding time of h19F6 or h42A5 is 180s, and the subsequent dissociation time is 1800 s; data were analyzed using a high-performance kinetic model, and only one concentration of wild-type FXI or chimeric FXI was detected in the SPR assay. The results show that both h-19F6 and h-42A5 bind to FXI and FXI/PK chimeras, except for the FXI/PK chimera in which the A3 domain is replaced by the corresponding domain of prekallikrein, suggesting that partial or complete epitopes of h-19F6 and h-42A5 are on the A3 domain.

Example 8: antibody functional neutralization of FXIa

The activity of human FXIa was determined primarily by measuring hydrolysis of a specific and fluorescently labeled substrate (S-2366, Diapharma Inc.). To test the inhibitory activity of the antibodies, the antibodies h-19F6, h-34F8 and h-42A5 to be tested were preincubated with FXIa in PBS buffer at a final concentration of 5nM for 5min at room temperature, then FXIa cleavage was initiated by addition of an equal volume of 1mM S-2366 and the change in absorbance over time was read continuously at 405nM using an M5e microplate reader (Molecular Devices Inc.), and the data were analyzed using GraphPad Prism software, as detailed in FIG. 4. The apparent Ki values of human antibodies h-19F6, h-34F8 and h-42A5 were 0.67, 2.08, 1.43 nM. Therefore, all three antibodies tested showed satisfactory inhibition of FXIa at relatively low concentrations.

Example 9: inhibition of FXIa-mediated FIX activation by antibodies

Experimental procedures for FXIa mediated FIX activation were performed as described above. anti-FXI antibodies can modulate the intrinsic pathway by inhibiting FXI activation and/or by inhibiting FXIa activity. First, the effect of two antibodies, h-19F6 and h-42A5, on FXIIa-mediated FXI activation was tested and it was found that neither h-19F6 nor h-42A5 prevented FXIIa-mediated conversion of FXI to FXIa (FIGS. 5C and 5D). The effect of these two antibodies on FXIa activity was then tested using FIX as substrate. As shown in FIGS. 5A and 5B, both h-19F6 and h-42A5 reduced FIX activation in a concentration-dependent manner. The inhibitory effect of these two antibodies on FXIa was further confirmed by the use of the chromogenic substrate S-2366 of FXIa. Both antibodies inhibited hydrolysis of S-2366 concentration-dependently (FIG. 4)

Example 10: evaluation of the Effect of anti-FXI antibodies on the clotting time of cynomolgus monkeys

To find animal species suitable for in vivo experiments, cross-reactivity of mouse, rat and monkey FXI antibodies was tested by APTT analysis. Antibodies prolonged APTT in monkey plasma, but not in mouse or rat plasmaExtension (data not shown). Therefore, prior to in vivo studies of thrombogenic potency, a monkey model was chosen to evaluate the pharmacodynamic effects of the three antibodies on clotting time. The cynomolgus monkey is injected with different antibodies to be tested with the appointed dose intravenously, superficial venous blood of upper limb is collected before administration and 0.5, 1, 3, 6, 12 and 24 hours after administration, and plasma anticoagulated by citric acid is prepared for determining APTT and PT. In APTT experiment, 50 μ l diluted plasma sample was taken, added with 25 μ l APTT reagent (SMN 10445709, Symen Inc.), mixed well, incubated at 37 ℃ for 4min, added with CaCl₂Solutions (25mM, SMN 10446232, Symen Inc.) were mixed in 25. mu.L and the time to thrombosis was recorded. In the PT assay, 50. mu.l of diluted plasma samples were taken, mixed with an equal volume of PT reagent (SMN 10446442, Symen Inc), and incubated at 37 ℃ for thrombogenesis time. The results show that 3 antibodies tested all prolonged APTT dose-dependently (see figure 6 for details) without affecting PT (see figure 7 for details).

Both h-19F6 and h-42A5 dose-dependently prolonged APTT (FIGS. 6B and 6C). Notably, at the same dose levels (0.3 and 1mg/kg), h-42A5 was more pronounced than h-19F6 in prolonging APTT, which is consistent with the in vitro effect of the antibody on human APTT (FIG. 16A). In addition, neither antibody affected PT in vivo (fig. 7B and 7C).

Example 11: effect of anti-FXI antibody on cynomolgus monkey arteriovenous shunt thrombosis and tail vein bleeding model

Different doses of the test antibody are administered to the same animal to assess thrombosis and bleeding time. The antibodies detected in this experiment were h-34F8, h-19F6, and h42A 5. Bleeding time and thrombosis were assessed in turn before and 30 minutes after each administration of antibody. 4 assessments were made for bleeding/thrombosis for three ascending doses (0.1, 0.3 and 1mg/kg) before and after dosing.

AV shunt thrombus assay: a weighed 10cm long silk thread is put into the tube, the cannula is connected with the femoral artery and the femoral vein, after the blood flow is released for 10min, the silk thread is quickly taken out, the wet weight is weighed, and the thrombus weight is calculated. Thrombus weight is determined by the difference in wire weighing before and after blood flow.

And (3) bleeding time measurement: a2 ml syringe was inserted into the tail vein of the animal and timing was started. The timing was stopped when the blood in the syringe no longer increased. This time period is the bleeding time.

The results show that all antibodies of the present invention can inhibit thrombus formation dose-dependently (see fig. 8)) without prolonging the time to tail bleeding (see fig. 9). The effect of h-19F6 and h-42A5 on thrombosis and hemostasis was evaluated in monkey models of AV shunt thrombosis and tail vein hemorrhage. Intravenous injection of h-19F6 resulted in a dose-dependent decrease in clot weight, and a significant decrease was observed at the 1mg/kg dose (fig. 8B). For the h-42a5 treated animals, thrombus weight was significantly reduced in a dose-dependent manner at all dose levels tested (fig. 8C). There was no significant change in bleeding time after treatment with h-19F6 or h-42A5 (FIGS. 9B and 9C).

Example 12: anti-FXI antibody on FeCl₃Effect of induced cynomolgus femoral artery thrombosis and Effect on Standard bleeding time

The cynomolgus monkeys were anesthetized with 1.5mg/kg Zoletil, intubated with trachea, ventilated, maintained for anesthesia with isoflurane, and monitored for blood pressure, heart rate and body temperature throughout. In the application of FeCl₃The first 2 hours of intravenous administration of test antibodies h-34F8, h-19F6, h-42A5 or blank vehicle (control). The left femoral artery was exposed and isolated using a blunt dissection method. Arterial blood flow was continuously detected and recorded with a Doppler flow probe. In the application of FeCl₃Previously, blood flow has been measured for at least 5 minutes. Then taking 2 pieces of FeCl₃Wrapping the surface of the artery above the Doppler flow probe with soaked filter paper, removing the filter paper after 10min, washing the wound with normal saline, and removing the residual FeCl₃The solution is washed clean. Blood flow was monitored throughout until the value dropped to 0, and 80% of the occlusion (20% of blood flow to baseline) and 100% of the blood flow (0) were recorded. For the same animal, bleeding time was determined by standard bleeding time assay before and 1 hour after dosing.

With FeCl₃Evaluation of induced arterial thrombosis with antibodies h-34F8, h-19F6 and h-42A5, i.e. 4 groups of monkeys were administered with air-white vehicle, h-34F8, h-19F6 and h-42A5, respectively, and 2 hours later, FeCl was applied to the left femoral artery₃Causing a thrombus and monitoring the blood flow velocity therebelow. The results show that: the blank control group has 80% blockage time and 100% blockage time of 14.66 +/-1.30 min and 18.5 +/-1.76 min respectively; 0.3mg/kg of h-34F8 and h-42A5, 80% of the occlusion time of the prevention and treatment group is 59.53 +/-16.95 min and 40.80 +/-7.94 min respectively, and 100% of the occlusion time is 70.40 +/-20.76 min and 50.61 +/-9.48 min respectively, which have significant difference compared with a blank control group (see figure 10); although the h-19F6 group showed no significant difference compared with the blank group, it also properly prolonged 80% occlusion (26.43 + -5.72 min) and 100% occlusion time (32.78 + -5.09 min) (see FIG. 10).

(Figure11D) the effect of the h-34F8, h-19F6 and h-42a5 antibodies on hemostasis was evaluated by the standard bleeding time method, and the results showed no significant difference in bleeding status between the groups before and 1 hour after administration (see figures 11A, 11B and 11C), and no significant difference in bleeding time between the h-34F8, h-19F6 and h-42a5 treated groups compared with the blank control group (see Figure11D).

The effect of the h-19F6 and h-42A5 antibodies on hemostasis was evaluated by a skin laceration induced hemorrhage test in which the test subjects were the same FeCl₃Induced arterial thrombosis (n-5 per group). Bleeding time was recorded before and 1 hour after dosing. No significant difference in bleeding time was observed between pre-and post-antibody administration 1 hour or between the three groups at 1 hour post-administration (fig. 18).

The effect of the antibody on in vitro clotting time of monkey plasma was also determined and the results showed that administration of 0.3mg/ml of the h-34F8, h-19F6, and h-42A5 antibodies significantly extended the Activated Partial Thromboplastin Time (APTT) to 3.29. + -. 0.20, 1.67. + -. 0.09, and 2.87. + -. 0.10 fold (FIG. 12A) before administration, but did not affect the Prothrombin Time (PT) (FIG. 12B), compared to the blank control (which did not cause prolongation of APTT).

Thus, the antibodies disclosed herein are effective in inhibiting the intrinsic pathway of coagulation without unexpectedly increasing bleeding time.

Example 13: evaluation of the Effect of modified anti-FXI antibodies on clotting time of cynomolgus monkeys for extended periods of time

The effect of two additional CMC optimized humanized anti-FXI antibodies ("modified h-19F 6" and "modified h-42a 5", respectively, as shown in figures 14 and 15) whose heavy and light chain sequences are listed in table 3 on clotting time in cynomolgus monkeys for extended periods of time (up to 14 days) was evaluated by APTT and PT assays as described in example 10. Cynomolgus monkeys were injected intravenously with 0.6mg/kg or 2.0mg/kg of the test antibody. Blood was collected from the upper limb superficial vein at 0.5 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 96 hours, 168 hours, 240 hours, and 336 hours before and after administration. As shown in fig. 14, both of the modified antibodies tested showed dose-dependent prolongation of APTT, as shown in fig. 15, neither of which affected PT. Both antibodies show prolonged therapeutic effect over a long period of time, which may be up to 7 days, up to 10 days, or up to 14 days.

Thus, it was unexpectedly found that the modified antibodies disclosed herein effectively inhibit the endogenous coagulation pathway over an extended period of time (up to 14 days) without showing any adverse effects of prolonged bleeding.

Example 14: effect on clotting time of Standard human plasma

Antibodies h-19F6 and h-42a5 were added to normal human plasma, and then APTT (fig. 16A) and PT (fig. 16B) (N ═ 3) were determined. Both the h-19F6 and h-42A5 antibodies extended the Activated Partial Thromboplastin Time (APTT) in standard human plasma in a concentration-dependent manner (FIG. 16A). Based on the established correlation curve between plasma FXI levels and APTT (data not shown), the maximal inhibition levels of FXI activity in plasma by h-19F6 and h-42A5 were about 97% and 99.5%, respectively. Neither antibody affected PT in human plasma (fig. 16B).

Example 15: binding characteristics of h-19F6 and h-42A5 to FXI

First, when h-19F6 and h-42A5 reacted with FXI in standard human plasma, their binding specificity was verified, and no reaction was detected in human FXI-poor plasma (FIG. 17). Biotinylated test antibodies were incubated with human normal plasma or FXI-depleted human plasma. The FXI antibody complex in plasma was eluted and western blotting was performed using mouse anti-human FXI IgG as the primary antibody. In Western blotting, 10. mu.L of human standard plasma or spent FXI plasma was used as FXI positive and FXI negativeAnd (4) performing positive control. Previously reported anti-FXI antibody 14E11¹⁷Shows the same binding profile as both antibodies (fig. 17).

The affinity of h-19F6 and h-42A5 for FXI was determined using Surface Plasmon Resonance (SPR) techniques. The antibody under test was captured on the sensor chip and then FXI at the indicated concentration was flowed through the chip. Sensorgrams for h-19F6 (FIG. 19A) and h-42A5 (FIG. 19B) were obtained. Dissociation constants for h-19F6 and h-42A5 were 22 and 36pM, respectively (FIGS. 19A and 19B).

The binding sites of these two antibodies on FXI were then determined. FXI is a homodimer consisting of 4 tandem Apple domains (a1-4) and a catalytic domain. Four mutants of FXI were generated by replacing each Apple domain with the corresponding domain of human prekallikrein, and tested for binding properties of h-19F6 or h-42A5 to the four mutants of FXI using SPR. An equal number of 4 mutant FXIs (where the a1, a2, A3 or a4 domains were replaced with the corresponding domains of prokinase-releasing enzyme) were immobilized on the sensor chip and the antibody to be tested (5 μ g/mL) was allowed to flow through the chip for binding. The amount of each antibody captured was recorded. Experiments were performed twice and representative results are described. Unexpectedly, both antibodies bound primarily to the A3 domain of FXI, as substitution of the A3 domain resulted in a greater reduction in binding of either antibody compared to substitution of the other 3 Apple domains (fig. 19C). In line with previous studies, another antibody, O1a6, is a known anti-FXI antibody, used as a positive control, that also binds specifically to the A3 domain of FXI.²¹However, it is speculated that h-19F6 and h-42A5 bind to different sites of FXI, since they have comparable affinity to FXI, but differ greatly in their inhibitory activity against FXI activity (FIG. 16). This hypothesis was tested by epitope binding using the Biacore T200 system. Indeed, binding of h-19F6 to FXI did not prevent further binding of h-42A5 to FXI, indicating that these two antibodies bound to different sites in the A3 domain of FXI (FIG. 19D). Then, the order of flow of the two antibodies was changed and it was found that h-42A5 binding to FXI did not prevent further binding of h-19F6 to FXI (data not shown).

Example 16: binding characteristics of h-19F6 and h-42A5 to FXIa

The antibody bound FXIa with good affinity to FXI (fig. 20A and 20B). The affinity of h-19F6 and h-42A5 for FXIa was determined using Surface Plasmon Resonance (SPR) techniques. Dissociation constants for h-19F6 and h-42A5 were 26 and 81pM, respectively (FIGS. 20A and 20B). The antibody under test was captured on the sensor chip and a specified concentration of FXIa was then flowed through the chip. Sensorgrams for h-19F6 (FIG. 20A) and h-42A5 (FIG. 20B) were obtained.

Reference to the literature

The references, patents and published patent applications listed below, and all references cited in the foregoing specification are hereby incorporated by reference in their entirety as if fully set forth herein.

1 Raskob,G.E.et al.Thrombosis:a major contributor to global disease burden. Arterioscler Thromb VascBiol 34,2363-2371(2014).

2 Gomez-Outes,A.,Garcia-Fuentes,M.&Suarez-Gea,M.L.Discovery methods of coagulation-inhibiting drugs.Expert Opin Drug Discov 12,1195-1205(2017).

3 Weitz,J.I.&Fredenburgh,J.C.Factors XI and XII as Targets for New Anticoagulants.Front Med(Lausanne)4,19(2017).

4 Muller,F.,Gailani,D.&Renne,T.Factor XI and XII as antithrombotic targets.Curr Opin Hematol 18,349-355(2011).

5 Al-Horani,R.A.&Desai,U.R.Factor XIa inhibitors:A review of the patent literature.Expert Opin Ther Pat 26,323-345(2016).

6 Chen,Z.,Seiffert,D.&Hawes,B.Inhibition of Factor XI activity as a promising antithrombotic strategy.Drug Discov Today 19,1435-1439(2014).

7 Gailani,D.&Gruber,A.Factor XI as a Therapeutic Target.Arterioscler Thromb Vasc Biol 36,1316-1322(2016).

8 Puy,C.,Rigg,R.A.&McCarty,O.J.The hemostatic role of factor XI.Thromb Res 141Suppl 2,S8-S11(2016).

9 Seligsohn,U.Factor XI deficiency in humans.J Thromb Haemost 7Suppl 1,84-87 (2009).

10 Preis,M.et al.Factor XI deficiency is associated with lower risk for cardiovascular and venous thromboembolism events.Blood 129,1210-1215(2017).

11 Meijers J.C.,Tekelenburg W.L.,Bouma B.N.,Bertina R.M.,Rosendaal F.R.High levels of coagulation factor XI as a risk factor for venous thrombosis.N Engl J Med 342, 696–701(2000).

12 Peyvandi,F.et al.Coagulation factor activity and clinical bleeding severity in rare bleeding disorders:results from the European Network of Rare Bleeding Disorders.J Thromb Haemost 10,615-621(2012).

13 Salomon,O.&Seligsohn,U.New observations on factor XI deficiency.Haemophilia 10 Suppl 4,184-187(2004).

14 Wang,X.et al.Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice.J Thromb Haemost 3,695-702(2005).

15 Wang,X.et al.Inhibition of Factor XIa Reduces the Frequency of Cerebral Microembolic Signals Derived from Carotid Arterial Thrombosis in Rabbits.J Pharmacol Exp Ther 360,476-483(2017).

16 Wong,P.C.,Crain,E.J.,Watson,C.A.&Schumacher,W.A.A small-molecule factor XIa inhibitor produces antithrombotic efficacy with minimal bleeding time prolongation in rabbits.J Thromb Thrombolysis32,129-137(2011).

17 Cheng,Q.et al.A role for factor XIIa-mediated factor XI activation in thrombus formation in vivo.Blood 116,3981-3989(2010).

18 Takahashi,M.et al.Inhibition of factor XI reduces thrombus formation in rabbit jugular vein under endothelial denudation and/or blood stasis.Thromb Res 125,464-470(2010).

19 Yau,J.W.et al.Selective depletion of factor XI or factor XII with antisense oligonucleotides attenuates catheter thrombosis in rabbits.Blood 123,2102-2107(2014).

20 Crosby,J.R.et al.Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates.Arterioscler Thromb Vasc Biol 33,1670-1678(2013).

21 Kravtsov D.V.et al.Factor XI contributes to thrombin generation in the absence of factor XII.Blood 114,452-458(2009).

22 Wang,X.et al.Effects of factor XI deficiency on ferric chloride-induced vena cava thrombosis in mice.J Thromb Haemost 4,1982-1988(2006).

23 Kleinschnitz,C.et al.Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis.J Exp Med 203,513-518(2006).

24 Gailani,D.,Lasky,N.M.&Broze,G.J.,Jr.A murine model of factor XI deficiency. Blood Coagul Fibrinolysis 8,134-144(1997).

25 Beck,A.,Wurch,T.,Bailly,C.&Corvaia,N.Strategies and challenges for the next generation of therapeutic antibodies.Nat Rev Immunol 10,345-352(2010).

26 Tucker,E.I.et al.Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI.Blood 113,936-944(2009).

27 Younis,H.S.et al.Antisense inhibition of coagulation factor XI prolongs APTT without increased bleeding risk in cynomolgus monkeys.Blood 119,2401-2408(2012).

28 Kouyama S,O.T.,Hagio T,et al.Discovery of ONO-5450598,a highly orally bioavailable small molecule factor XIa inhibitor:the pharmacokinetic and pharmacological profiles.Res Pract Thromb Haemost 1 Suppl 1:PB 2139(2017).

29 Wong,P.C.et al.In vitro,antithrombotic and bleeding time studies of BMS- 654457,a small-molecule,reversible and direct inhibitor of factor XIa.J Thromb Thrombolysis 40,416-423(2015).

30 David,T.et al.Factor XIa-specific IgG and a reversal agent to probe factor XI function in thrombosis and hemostasis.Sci Transl Med 8,353ra112(2016).

31 van Montfoort,M.L.et al.Two novel inhibitory anti-human factor XI antibodies prevent cessation of blood flow in a murine venous thrombosis model.Thromb Haemost 110,1065-1073(2013).

32 Buller,H.R.et al.Factor XI antisense oligonucleotide for prevention of venous thrombosis.N Engl J Med 372,232-240(2015).

33 Emsley et al.,Blood 115(13):2569-2577(2010).

34 Wang et al.,J.Pharm.Sciences 96(1):1-26(2007).

SEQUENCE LISTING

<110> Shanghai Rencui biopharmaceutical GmbH

<120> anti-coagulation factor XI antibodies

<130> 057783-8013.CN02

<140> PCT/CN2018/099638

<141> 2018-08-09

<160> 209

<170> PatentIn version 3.5

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gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca gagccagcga aagtgttgat aattatgcca ttagttttat gaactggttc 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa cctaggatcc 180

ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240

cctatggagg aggatgatac tgcaatgtat ttctgtcagc aagataagga ggttccgtgg 300

acgttcggtg gaggcaccga gctggaaatc aaa 333

<210> 2

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-VH

<400> 2

caggtcactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

acttgttctt tctctgggtt ttcactgaac actcctggta tgggtgtgag ctggattcgt 120

cagccttcag gaaagggtct ggaatggctg gcacacattt actgggatga tgacaagcgc 180

tttaacccat ccctgaagag ccgactcaca atctccaagg atacctccag agatcaggta 240

ttcctcatga tcaccagtgt ggacactgca gattctgcca catacttctg tgctcgaaaa 300

ggccgcgggc cctttactta ctggggccaa gggactctgg tcactgtctc ttca 354

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<211> 45

<212> DNA

<213> Artificial Sequence

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<223> Synthetic: 3G12-CDR-L1

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agagccagcg aaagtgttga taattatgcc attagtttta tgaac 45

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence

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<223> Synthetic: 3G12-CDR-L2

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gctgcatcca acctaggatc c 21

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<211> 27

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<223> Synthetic: 3G12-CDR-L3

<400> 5

cagcaagata aggaggttcc gtggacg 27

<210> 6

<211> 21

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<223> Synthetic: 3G12-CDR-H1

<400> 6

actcctggta tgggtgtgag c 21

<210> 7

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-CDR-H2

<400> 7

cacatttact gggatgatga caagcgcttt aacccatccc tgaagagc 48

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-CDR-H3

<400> 8

aaaggccgcg ggccctttac ttac 24

<210> 9

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-VL

<400> 9

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

1 5 10 15

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

20 25 30

Ala Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Asp Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys

100 105 110

<210> 10

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-VH

<400> 10

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Asn Thr Pro

20 25 30

Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Phe Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg Asp Gln Val

65 70 75 80

Phe Leu Met Ile Thr Ser Val Asp Thr Ala Asp Ser Ala Thr Tyr Phe

85 90 95

Cys Ala Arg Lys Gly Arg Gly Pro Phe Thr Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 11

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-CDR-L1

<400> 11

Arg Ala Ser Glu Ser Val Asp Asn Tyr Ala Ile Ser Phe Met Asn

1 5 10 15

<210> 12

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-CDR-L2

<400> 12

Ala Ala Ser Asn Leu Gly Ser

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-CDR-L3

<400> 13

Gln Gln Asp Lys Glu Val Pro Trp Thr

1 5

<210> 14

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-CDR-H1

<400> 14

Thr Pro Gly Met Gly Val Ser

1 5

<210> 15

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-CDR-H2

<400> 15

His Ile Tyr Trp Asp Asp Asp Lys Arg Phe Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 3G12-CDR-H3

<400> 16

Lys Gly Arg Gly Pro Phe Thr Tyr

1 5

<210> 17

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-VL

<400> 17

gacattgtgc tgacccaatc tccagcctct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca gagccagcga aagtgttgat aattatggca ttagttttct gaactggttc 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa tctaggatcc 180

ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240

cctatggagg aggatgatac tgcaatgtat ttctgtcagc aagataaggg ggttccgtgg 300

acgttcggtg gaggcaccaa gctggaaatg aaa 333

<210> 18

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-VH

<400> 18

caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

acttgttctt tctctgggtt ttcactgaac acttctggta tgggtgtgag ctggattcgt 120

cagccttcag gaaagggtct ggagtggctg gcacacattt actgggatga tgacaagcgc 180

tataaaccat ccctgaagag ccggctcaca atctccaagg atacctccag aaaccaggta 240

ttcctcatga tcaccagtgt ggacactgca gatactgcca catactactg tgttcgaaaa 300

ggccgcgggc cctttgctaa ctggggccaa gggactctgg tcactgtctc tgca 354

<210> 19

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-L1

<400> 19

agagccagcg aaagtgttga taattatggc attagttttc tgaac 45

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-L2

<400> 20

gctgcatcca atctaggatc c 21

<210> 21

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-L3

<400> 21

cagcaagata agggggttcc gtggacg 27

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-H1

<400> 22

acttctggta tgggtgtgag c 21

<210> 23

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-H2

<400> 23

cacatttact gggatgatga caagcgctat aaaccatccc tgaagagc 48

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-H3

<400> 24

aaaggccgcg ggccctttgc taac 24

<210> 25

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-VL

<400> 25

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

1 5 10 15

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

20 25 30

Gly Ile Ser Phe Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Asp Lys

85 90 95

Gly Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys

100 105 110

<210> 26

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-VH

<400> 26

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Asn Thr Ser

20 25 30

Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Lys Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val

65 70 75 80

Phe Leu Met Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Val Arg Lys Gly Arg Gly Pro Phe Ala Asn Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 27

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-L1

<400> 27

Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Leu Asn

1 5 10 15

<210> 28

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-L2

<400> 28

Ala Ala Ser Asn Leu Gly Ser

1 5

<210> 29

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-L3

<400> 29

Gln Gln Asp Lys Gly Val Pro Trp Thr

1 5

<210> 30

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-H1

<400> 30

Thr Ser Gly Met Gly Val Ser

1 5

<210> 31

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-H2

<400> 31

His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Lys Pro Ser Leu Lys Ser

1 5 10 15

<210> 32

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 5B2-CDR-H3

<400> 32

Lys Gly Arg Gly Pro Phe Ala Asn

1 5

<210> 33

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-VL

<400> 33

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattgat attcgcttaa actggcttcg acaggaacca 120

gatggaacta ttaaacgcct gatctacgcc acatccagtt tagattctgg tgtccccaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg ttgactatta ctgtctacaa tatgctagtt ctccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 34

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-VH

<400> 34

cagatccagt tggtgcagtc tggacctgaa ctgaagaagc ctggagagac cgtcaagatc 60

tcctgcaagg cttctgggta tattttcaca gactatggaa tgaactgggt gaagcaggct 120

ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactggaga gccaacatat 180

gctgatgact tcaagggacg gtttgtcttc tctttggaaa cctctgccag cactgcctat 240

ttacagatca acaacctcaa aaatgaggac acggctacat ttttctgtgc aagaaggagg 300

atgggttatg ctgtggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354

<210> 35

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-L1

<400> 35

cgggcaagtc aggacattga tattcgctta aac 33

<210> 36

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-L2

<400> 36

gccacatcca gtttagattc t 21

<210> 37

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-L3

<400> 37

ctacaatatg ctagttctcc attcacg 27

<210> 38

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-H1

<400> 38

gactatggaa tgaac 15

<210> 39

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-H2

<400> 39

tggataaaca cctacactgg agagccaaca tatgctgatg acttcaaggg a 51

<210> 40

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-H3

<400> 40

aggaggatgg gttatgctgt ggactac 27

<210> 41

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-VL

<400> 41

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

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Asp Ile Arg

20 25 30

Leu Asn Trp Leu Arg Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 42

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-VH

<400> 42

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

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

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

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Phe Phe Cys

85 90 95

Ala Arg Arg Arg Met Gly Tyr Ala Val Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 43

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-L1

<400> 43

Arg Ala Ser Gln Asp Ile Asp Ile Arg Leu Asn

1 5 10

<210> 44

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-L2

<400> 44

Ala Thr Ser Ser Leu Asp Ser

1 5

<210> 45

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-L3

<400> 45

Leu Gln Tyr Ala Ser Ser Pro Phe Thr

1 5

<210> 46

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-H1

<400> 46

Asp Tyr Gly Met Asn

1 5

<210> 47

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-H2

<400> 47

Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys

1 5 10 15

Gly

<210> 48

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7C9-CDR-H3

<400> 48

Arg Arg Met Gly Tyr Ala Val Asp Tyr

1 5

<210> 49

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-VL

<400> 49

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca gagccagcga aagtgttgat aattatgcca ttagttttat gaattggttc 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa cctaggatcc 180

ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240

cctatggagg aggatgatac tgcaatgtat ttctgtcagc aagataagga ggttccgtgg 300

acgttcggtg gaggcaccaa gctggagctg aaa 333

<210> 50

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-VH

<400> 50

caggttactc tgaaagagtc tggccctggg atagtgcagc cctcccagac cctcaatctg 60

acttgttctt tctctggatt ttcactgagc acttctggta tgggtgtgag ctggattcgt 120

cagccttcag gaaagggtct ggattggctg gcacacattt actgggatga tgacaagcgc 180

tataacccat ccctgatgag ccggctcaca atctccaagg atacctccag aaaccaggta 240

ttcctcatga tcaccagtgt ggacactgca gatactgcca catactactg tgctcgaaaa 300

ggccgcgggc cctttgctta ctggggccaa gggactctgg tcactgtctc ttca 354

<210> 51

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-L1

<400> 51

agagccagcg aaagtgttga taattatgcc attagtttta tgaat 45

<210> 52

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-L2

<400> 52

gctgcatcca acctaggatc c 21

<210> 53

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-L3

<400> 53

cagcaagata aggaggttcc gtggacg 27

<210> 54

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-H1

<400> 54

acttctggta tgggtgtgag c 21

<210> 55

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-H2

<400> 55

cacatttact gggatgatga caagcgctat aacccatccc tgatgagc 48

<210> 56

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-H3

<400> 56

aaaggccgcg ggccctttgc ttac 24

<210> 57

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-VL

<400> 57

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

1 5 10 15

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

20 25 30

Ala Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Asp Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 58

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-VH

<400> 58

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

1 5 10 15

Thr Leu Asn Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Asp

35 40 45

Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Phe Leu Met Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Lys Gly Arg Gly Pro Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 59

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-L1

<400> 59

Arg Ala Ser Glu Ser Val Asp Asn Tyr Ala Ile Ser Phe Met Asn

1 5 10 15

<210> 60

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-L2

<400> 60

Ala Ala Ser Asn Leu Gly Ser

1 5

<210> 61

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-L3

<400> 61

Gln Gln Asp Lys Glu Val Pro Trp Thr

1 5

<210> 62

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-H1

<400> 62

Thr Ser Gly Met Gly Val Ser

1 5

<210> 63

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-H2

<400> 63

His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Met Ser

1 5 10 15

<210> 64

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 7F1-CDR-H3

<400> 64

Lys Gly Arg Gly Pro Phe Ala Tyr

1 5

<210> 65

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-VL

<400> 65

cagttcacgc tgactcaacc aaagtccgtg tcaggatctt taagaagcac tatcaccatt 60

ccctgtgagc gcagcagtgg tgacattgga gatagctatg tgagctggta ccaacaacac 120

ttgggaagac cccccatcaa tgtgatctat gctgatgatc aaagaccatc tgaagtgtct 180

gctcggttct cgggctccat cgacagctcc tctaactcag cctcactgac catcactaat 240

ctacagatgg atgatgaggc cgactacttc tgtcagtctt acgatactta tatggatgtt 300

gtgttcggtg gtggaaccaa gctcaatgtc cta 333

<210> 66

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-VH

<400> 66

gaggtgcagc tgaaggaatc aggacctggt ctggtgcagc cctcacagac cctgtccctc 60

acctgcactg tctctggatt ctcattaacg gactacagtg tacactgggt tcgccagcct 120

ccaggaaaag gtctggagtg gatgggagta atgtggagtg gtggaagcac agcatataat 180

ccagctctca catcccgact gaccattagc agggacacct ccaagagcca agttttctta 240

aaaatgaaca gtctgcaaac tgaagataca gccatttact actgtaccag agcacctttt 300

aacaactggg gcaattggct tccttactgg ggccaaggca ctctggtcac tgtctcttca 360

<210> 67

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-L1

<400> 67

gagcgcagca gtggtgacat tggagatagc tatgtgagc 39

<210> 68

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-L2

<400> 68

gctgatgatc aaagaccatc t 21

<210> 69

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-L3

<400> 69

cagtcttacg atacttatat ggatgttgtg 30

<210> 70

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-H1

<400> 70

gactacagtg tacac 15

<210> 71

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-H2

<400> 71

gtaatgtgga gtggtggaag cacagcatat aatccagctc tcacatcc 48

<210> 72

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-H3

<400> 72

gcacctttta acaactgggg caattggctt ccttac 36

<210> 73

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-VL

<400> 73

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

1 5 10 15

Thr Ile Thr Ile Pro Cys Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser

20 25 30

Tyr Val Ser Trp Tyr Gln Gln His Leu Gly Arg Pro Pro Ile Asn Val

35 40 45

Ile Tyr Ala Asp Asp Gln Arg Pro Ser Glu Val Ser Ala Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Thr Asn

65 70 75 80

Leu Gln Met Asp Asp Glu Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Thr

85 90 95

Tyr Met Asp Val Val Phe Gly Gly Gly Thr Lys Leu Asn Val Leu

100 105 110

<210> 74

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-VH

<400> 74

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Met Trp Ser Gly Gly Ser Thr Ala Tyr Asn Pro Ala Leu Thr

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Thr

85 90 95

Arg Ala Pro Phe Asn Asn Trp Gly Asn Trp Leu Pro Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 75

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-L1

<400> 75

Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser Tyr Val Ser

1 5 10

<210> 76

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-L2

<400> 76

Ala Asp Asp Gln Arg Pro Ser

1 5

<210> 77

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-L3

<400> 77

Gln Ser Tyr Asp Thr Tyr Met Asp Val Val

1 5 10

<210> 78

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-H1

<400> 78

Asp Tyr Ser Val His

1 5

<210> 79

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-H2

<400> 79

Val Met Trp Ser Gly Gly Ser Thr Ala Tyr Asn Pro Ala Leu Thr Ser

1 5 10 15

<210> 80

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 13F4-CDR-H3

<400> 80

Ala Pro Phe Asn Asn Trp Gly Asn Trp Leu Pro Tyr

1 5 10

<210> 81

<211> 339

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-VL

<400> 81

caattcacgc tgactcaacc aaagtccgtg tcaggctctt taagaagcac tatcaccatt 60

ccctgtgagc gcagcagtgg tgacattgga gatagctatg tgagctggta ccagcaacac 120

ttgggaagac cccccatcaa tgtgatctat gctgatgatc aaagaccatc tgaagtgtct 180

gatcggttct cgggctccat cgacacctcc tctaactcag cctcactgac catcactaat 240

ctgcagatgg atgatgcggc cgactacttc tgtcagtctt acgatagtaa tattgatttt 300

aaccctgttt tcggtggtgg aaccaagctc actgtccta 339

<210> 82

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-VH

<400> 82

gaggtgcagc tggtggagtc tggtggaggc ttagtgcagc ctggaaggtc tctgagactc 60

tcctgtacag cctcaggatt cactttcagt aaatatgtca tggcctgggt ccgccaggct 120

ccaacgaagg ggctggagtg ggtcgcatcc attaattatg atggtagtac cacttactat 180

cgagactccg tgcagggccg gttcactctc tccagagata atgcaaaaac caccctatac 240

ctgcaaatgg acagtctgag gtctgaggac acggccactt attactgtgc aaggcaccct 300

tttaacaact tcgggatttg gtttgcttac tggggccaag gcactctggt cactgtctct 360

tca 363

<210> 83

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-L1

<400> 83

gagcgcagca gtggtgacat tggagatagc tatgtgagc 39

<210> 84

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-L2

<400> 84

gctgatgatc aaagaccatc t 21

<210> 85

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-L3

<400> 85

cagtcttacg atagtaatat tgattttaac cctgtt 36

<210> 86

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-H1

<400> 86

aaatatgtca tggcc 15

<210> 87

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-H2

<400> 87

tccattaatt atgatggtag taccacttac tatcgagact ccgtgcaggg c 51

<210> 88

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-H3

<400> 88

caccctttta acaacttcgg gatttggttt gcttac 36

<210> 89

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-VL

<400> 89

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

1 5 10 15

Thr Ile Thr Ile Pro Cys Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser

20 25 30

Tyr Val Ser Trp Tyr Gln Gln His Leu Gly Arg Pro Pro Ile Asn Val

35 40 45

Ile Tyr Ala Asp Asp Gln Arg Pro Ser Glu Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Thr Ser Ser Asn Ser Ala Ser Leu Thr Ile Thr Asn

65 70 75 80

Leu Gln Met Asp Asp Ala Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Ser

85 90 95

Asn Ile Asp Phe Asn Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val

100 105 110

Leu

<210> 90

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-VH

<400> 90

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Val Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Gln Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg His Pro Phe Asn Asn Phe Gly Ile Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 91

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-L1

<400> 91

Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser Tyr Val Ser

1 5 10

<210> 92

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-L2

<400> 92

Ala Asp Asp Gln Arg Pro Ser

1 5

<210> 93

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-L3

<400> 93

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

1 5 10

<210> 94

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-H1

<400> 94

Lys Tyr Val Met Ala

1 5

<210> 95

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-H2

<400> 95

Ser Ile Asn Tyr Asp Gly Ser Thr Thr Tyr Tyr Arg Asp Ser Val Gln

1 5 10 15

Gly

<210> 96

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 19F6-CDR-H3

<400> 96

His Pro Phe Asn Asn Phe Gly Ile Trp Phe Ala Tyr

1 5 10

<210> 97

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-VL

<400> 97

gatgtccgga tgacacagtc tccagcttcc ctgtctgcat ctctgggaga aactgtcaac 60

atcgaatgtc tagcaagtga ggacatttac agtgatttag catggtatca gcagaagcca 120

gggaaatctc ctcagctcct gatctataat gcaaatagtc tacaaaatgg ggtcccttca 180

cggtttagtg gcagtggttc tggcacgcag tattctctaa aaatatccac cctgcaatct 240

gaagatgtcg cgacttattt ctgtcaacaa tatagcaatt atcgtcggac gttcggtgga 300

ggcaccaagc tggaaatcaa t 321

<210> 98

<211> 381

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-VH

<400> 98

gaggtgcaac tggtggagtc tgggggaggc ctagtgcagc ctggaaggtc tctgaaacta 60

tcctgtgtag cctctggatt cacattcaac aaccactgga tgacctggat ccgccaggct 120

ccagggaagg ggctggagtg ggttgcatcc attactgata atggtggtag cacttactat 180

ccagactctg tgaagggccg attcactatc tccagagata atgcaaaaag caccctatac 240

ctgcacatga acagtctgag gtctgaggac acggccactt attactgtac aagagatcgg 300

tatgactctg atggttatta ttacgtgagg tactatgttg tggacgcctg gggtcaagga 360

gcttcagtca ctgtctcctc a 381

<210> 99

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-L1

<400> 99

ctagcaagtg aggacattta cagtgattta gca 33

<210> 100

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-L2

<400> 100

aatgcaaata gtctacaaaa t 21

<210> 101

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-L3

<400> 101

caacaatata gcaattatcg tcggacg 27

<210> 102

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-H1

<400> 102

aaccactgga tgacc 15

<210> 103

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-H2

<400> 103

tccattactg ataatggtgg tagcacttac tatccagact ctgtgaaggg c 51

<210> 104

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-H3

<400> 104

gatcggtatg actctgatgg ttattattac gtgaggtact atgttgtgga cgcc 54

<210> 105

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-VL

<400> 105

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

1 5 10 15

Glu Thr Val Asn Ile Glu Cys Leu Ala Ser Glu Asp Ile Tyr Ser Asp

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Thr Leu Gln Ser

65 70 75 80

Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Asn Tyr Arg Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn

100 105

<210> 106

<211> 127

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-VH

<400> 106

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asn Asn His

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu His Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Arg Asp Arg Tyr Asp Ser Asp Gly Tyr Tyr Tyr Val Arg Tyr Tyr

100 105 110

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

115 120 125

<210> 107

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-L1

<400> 107

Leu Ala Ser Glu Asp Ile Tyr Ser Asp Leu Ala

1 5 10

<210> 108

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-L2

<400> 108

Asn Ala Asn Ser Leu Gln Asn

1 5

<210> 109

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-L3

<400> 109

Gln Gln Tyr Ser Asn Tyr Arg Arg Thr

1 5

<210> 110

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-H1

<400> 110

Asn His Trp Met Thr

1 5

<210> 111

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-H2

<400> 111

Ser Ile Thr Asp Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 112

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 21F12-CDR-H3

<400> 112

Asp Arg Tyr Asp Ser Asp Gly Tyr Tyr Tyr Val Arg Tyr Tyr Val Val

1 5 10 15

Asp Ala

<210> 113

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-VL

<400> 113

gatgttgtgt tgacacagac tccaggttcc ctgtctgtca cacttggaca gcaagtttct 60

atatcctgta ggtctagtca gagcctggaa agtcgtgatg ggaacactta tttggaatgg 120

tacctacaga agccaggcca gtctccacag gtcctcctct atggagtttc caaccgattg 180

tctggggtcc cagacaggtt ccttggcaga gggtcagggg cagatttcac cctcaagatc 240

agcagagtag agcctgagga cttgggagtt tattactgct tccaagctac acatggtcca 300

ttcacgttcg gctcagggac gaagttggaa atgaaa 336

<210> 114

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-VH

<400> 114

caggtgcagc tgaaggagtc aggacctggc ctggtgcagc cctcacagac cctgtctctc 60

acctgcactg tctctgggtt ctcattaacc acctatcatg tgcactgggt tcgacagcct 120

ccaggaaaag gtctggagtg gatgggaata atgtggagag atggagacac atcatataat 180

tcagttctca aatctcgact gagcatcagc agggacatct ccaagagcca agttttctta 240

aaaatgagca gtctgcaaac tgaagacaca gccacttact tctgtgccag aggggggact 300

cttacaactc cctttactta ctggggccaa ggcactctgg tcactgtctc ttca 354

<210> 115

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-L1

<400> 115

aggtctagtc agagcctgga aagtcgtgat gggaacactt atttggaa 48

<210> 116

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-L2

<400> 116

ggagtttcca accgattgtc t 21

<210> 117

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-L3

<400> 117

ttccaagcta cacatggtcc attcacg 27

<210> 118

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-H1

<400> 118

acctatcatg tgcac 15

<210> 119

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-H2

<400> 119

ataatgtgga gagatggaga cacatcatat aattcagttc tcaaatct 48

<210> 120

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-H3

<400> 120

ggggggactc ttacaactcc ctttacttac 30

<210> 121

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-VL

<400> 121

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

1 5 10 15

Gln Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Ser Arg

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Val Leu Leu Tyr Gly Val Ser Asn Arg Leu Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Pro Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

Thr His Gly Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Met Lys

100 105 110

<210> 122

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-VH

<400> 122

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

His Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Met Trp Arg Asp Gly Asp Thr Ser Tyr Asn Ser Val Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Arg Asp Ile Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Ser Ser Leu Gln Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala

85 90 95

Arg Gly Gly Thr Leu Thr Thr Pro Phe Thr Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 123

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-L1

<400> 123

Arg Ser Ser Gln Ser Leu Glu Ser Arg Asp Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 124

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-L2

<400> 124

Gly Val Ser Asn Arg Leu Ser

1 5

<210> 125

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-L3

<400> 125

Phe Gln Ala Thr His Gly Pro Phe Thr

1 5

<210> 126

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-H1

<400> 126

Thr Tyr His Val His

1 5

<210> 127

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-H2

<400> 127

Ile Met Trp Arg Asp Gly Asp Thr Ser Tyr Asn Ser Val Leu Lys Ser

1 5 10 15

<210> 128

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 34F8-CDR-H3

<400> 128

Gly Gly Thr Leu Thr Thr Pro Phe Thr Tyr

1 5 10

<210> 129

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-VL

<400> 129

gatatccgga tgacacagtc tccagcttcc ctgtctgcat ctctgggaga aactgtcaac 60

atcgaatgtc tagcaagtga ggacatttac agtgatttag catggtatca gcagaagcca 120

gggaaatctc cacaactcct gatctataat gcaaatagcg tgcaaaatgg ggtcccttca 180

cggtttagtg gcagtggatc tggcacacag tattctctaa aaataaacag cctgcaatct 240

gaagatgtcg cgacttattt ctgtcaacag tttaacagtt atccgaacac gtttggagct 300

gggaccaagc tggaaatcaa a 321

<210> 130

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-VH

<400> 130

gaggtgcaac ttcaggagtc aggacctggc cttgtgaaac cctcacagtc actctccctc 60

acctgttctg tctctggttt ctccatcact aataattact ggggctggat ccggaagttc 120

ccaagaaata aaatggagtg gattggacac ataagctaca gtggtagcac taactacaac 180

ccatctctca aaagtcgcat ctccattact agagactcat cgaagagtca gttcttcctg 240

cagttgaact ctttaactac tgaggacaca gccacatatt actgtgcaag aggatcttat 300

tactatagcg catcgggcta ctttgattat tggggccaag gaatcacggt cacagtctcc 360

tca 363

<210> 131

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-L1

<400> 131

ctagcaagtg aggacattta cagtgattta gca 33

<210> 132

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-L2

<400> 132

aatgcaaata gcgtgcaaaa t 21

<210> 133

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-L3

<400> 133

caacagttta acagttatcc gaacacg 27

<210> 134

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-H1

<400> 134

aataattact ggggc 15

<210> 135

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-H2

<400> 135

cacataagct acagtggtag cactaactac aacccatctc tcaaaagt 48

<210> 136

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-H3

<400> 136

ggatcttatt actatagcgc atcgggctac tttgattat 39

<210> 137

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-VL

<400> 137

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

1 5 10 15

Glu Thr Val Asn Ile Glu Cys Leu Ala Ser Glu Asp Ile Tyr Ser Asp

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 138

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-VH

<400> 138

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Ser Gly Phe Ser Ile Thr Asn Asn

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Phe Pro Arg Asn Lys Met Glu Trp Ile

35 40 45

Gly His Ile Ser Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gly Ser Tyr Tyr Tyr Ser Ala Ser Gly Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Ile Thr Val Thr Val Ser Ser

115 120

<210> 139

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-L1

<400> 139

Leu Ala Ser Glu Asp Ile Tyr Ser Asp Leu Ala

1 5 10

<210> 140

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-L2

<400> 140

Asn Ala Asn Ser Val Gln Asn

1 5

<210> 141

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-L3

<400> 141

Gln Gln Phe Asn Ser Tyr Pro Asn Thr

1 5

<210> 142

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-H1

<400> 142

Asn Asn Tyr Trp Gly

1 5

<210> 143

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-H2

<400> 143

His Ile Ser Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 144

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 38E4-CDR-H3

<400> 144

Gly Ser Tyr Tyr Tyr Ser Ala Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 145

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-VL

<400> 145

gacgtggtct tgacccaaac ccctggatca cttagcgtga cactgggcga tccagcatca 60

atgtcctgca gaagctccca gtccttggag agtagcgacg gcaacacata cctcgagtgg 120

tatctgcaga aatccgggca gtccccacag ctgctgatct acggcgtgag taacaggttc 180

agcggggtgc ctgataggtt cgccggcagc gggtccggga cagattttac tctcaagatt 240

agccgcgtcg aacccgagga cctgggcgtg tactactgtt ttcaggccac tcgggacccc 300

tttactttcg ggagcgggac aaagctggag attaat 336

<210> 146

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-VH

<400> 146

caggtccagc ttaaagagtc cggacctgga cttgtgcagc catcccagac cttgtccttg 60

acctgcaccg tgtcagggtt ctctctcacc agttaccacc tgcattggat caggcagcct 120

cccggcaagg ggctggaatg gatggggctg atgtggagag atggggatac atcttacaac 180

agcaggctga agagccggct gagcattaca cgggacacca gcaagtccca ggtgttcctc 240

aagatgagcg ggctccaaac tgaggacaca gctacatact actgtgcacg cggcatgaca 300

ctcgccactc cctttctgta ttggggccag ggcactctgg tcactgtgtc ctca 354

<210> 147

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-L1

<400> 147

agaagctccc agtccttgga gagtagcgac ggcaacacat acctcgag 48

<210> 148

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-L2

<400> 148

ggcgtgagta acaggttcag c 21

<210> 149

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-L3

<400> 149

tttcaggcca ctcgggaccc ctttact 27

<210> 150

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-H1

<400> 150

agttaccacc tgcat 15

<210> 151

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-H2

<400> 151

ctgatgtgga gagatgggga tacatcttac aacagcaggc tgaagagc 48

<210> 152

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-H3

<400> 152

ggcatgacac tcgccactcc ctttctgtat 30

<210> 153

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-VL

<400> 153

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

1 5 10 15

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

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Ser Gly Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

Ser Arg Val Glu Pro Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

Thr Arg Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn

100 105 110

<210> 154

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-VH

<400> 154

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

His Leu His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Leu Met Trp Arg Asp Gly Asp Thr Ser Tyr Asn Ser Arg Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Ser Gly Leu Gln Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Gly Met Thr Leu Ala Thr Pro Phe Leu Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 155

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-L1

<400> 155

Arg Ser Ser Gln Ser Leu Glu Ser Ser Asp Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 156

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-L2

<400> 156

Gly Val Ser Asn Arg Phe Ser

1 5

<210> 157

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-L3

<400> 157

Phe Gln Ala Thr Arg Asp Pro Phe Thr

1 5

<210> 158

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-H1

<400> 158

Ser Tyr His Leu His

1 5

<210> 159

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-H2

<400> 159

Leu Met Trp Arg Asp Gly Asp Thr Ser Tyr Asn Ser Arg Leu Lys Ser

1 5 10 15

<210> 160

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42A5-CDR-H3

<400> 160

Gly Met Thr Leu Ala Thr Pro Phe Leu Tyr

1 5 10

<210> 161

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-VL

<400> 161

gatatccgga tgacacagtc gccagcttcc ctgtctgcat ctctgggaga aactgtcaac 60

atcgaatgtc tagcaagtga ggacattcac agtgatttag catggtatca gcagaagcca 120

gggaaatctc ctcagctcct gatctataat gcaaatagct tgcaaaatgg ggtcccttca 180

cggttcagtg gcagtggatc tggcacacag tattctctaa aaataaccag cctgcaatct 240

gaagatgtcg cgacttattt ctgtcaacaa tataccaact atccgaacac gtttggagcg 300

gggaccaagc tggaaatcaa t 321

<210> 162

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-VH

<400> 162

gaggtgcagc ttcaggagtc aggacctggc cttgtgaaac cctcacagtc actctccctc 60

acctgttctg tcactggtta ctccatcact aatcattact ggggctggat ccggaaattc 120

ccaggaaata aaatggagtg gattggacac ataagcaaca gtggtggcac taactacaac 180

ccatcactca aaagtcgaat ctccattact agagacacat cgaagaatca gttcttcctg 240

cagttgaagt ctgtaactac tgaggacaca gccacatatt actgtacaag aggatcttat 300

tactatagcg catcgggcta ctttgattac tggggccaag gagtcctggt cacagtctcc 360

tcc 363

<210> 163

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-L1

<400> 163

ctagcaagtg aggacattca cagtgattta gca 33

<210> 164

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-L2

<400> 164

aatgcaaata gcttgcaaaa t 21

<210> 165

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-L3

<400> 165

caacaatata ccaactatcc gaacacg 27

<210> 166

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-H1

<400> 166

aatcattact ggggc 15

<210> 167

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-H2

<400> 167

cacataagca acagtggtgg cactaactac aacccatcac tcaaaagt 48

<210> 168

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-H3

<400> 168

ggatcttatt actatagcgc atcgggctac tttgattac 39

<210> 169

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-VL

<400> 169

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

1 5 10 15

Glu Thr Val Asn Ile Glu Cys Leu Ala Ser Glu Asp Ile His Ser Asp

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Leu Gln Ser

65 70 75 80

Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Thr Asn Tyr Pro Asn

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Asn

100 105

<210> 170

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-VH

<400> 170

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Asn His

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Ile

35 40 45

Gly His Ile Ser Asn Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu

65 70 75 80

Gln Leu Lys Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Thr

85 90 95

Arg Gly Ser Tyr Tyr Tyr Ser Ala Ser Gly Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Leu Val Thr Val Ser Ser

115 120

<210> 171

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-L1

<400> 171

Leu Ala Ser Glu Asp Ile His Ser Asp Leu Ala

1 5 10

<210> 172

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-L2

<400> 172

Asn Ala Asn Ser Leu Gln Asn

1 5

<210> 173

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-L3

<400> 173

Gln Gln Tyr Thr Asn Tyr Pro Asn Thr

1 5

<210> 174

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-H1

<400> 174

Asn His Tyr Trp Gly

1 5

<210> 175

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-H2

<400> 175

His Ile Ser Asn Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 176

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 42F4-CDR-H3

<400> 176

Gly Ser Tyr Tyr Tyr Ser Ala Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 177

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-VL

<400> 177

gatatccgga tgacacagtc tccagcttcc ctgtctgcat ctctgggaga aactgtcaac 60

atcggatgtc tagcaagtga ggacatttac agtgatttag catggtatca gcagaagcca 120

gggaagtctc ctcagctcct gatctataat gcaaataact tgcaaaatgg ggtcccttca 180

cggtttagtg gcagtggatc tggcacacaa tattctctaa aaataaacag cctgcaatct 240

gaagatgtcg cgacttattt ctgtcaacaa tataacagtt atccgaacac gtttggagct 300

gggaccaagc tggaaataaa a 321

<210> 178

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-VH

<400> 178

gaggtgcagc ttcaggagtc aggacctggc cttgtgaaac cctcacagtc actctccctc 60

atttgttctg tcactggtta ctccatcact acaacttact ggggctggat ccggaagttc 120

ccaggaaata aaatggagtg gattggacac ataagtaaca gtggtagtac taattacaac 180

ccatctctca aaagtcgaat ctccgttact agagacacat cgacgaatca gttcttcctg 240

cagttgaact ctgtaactac tgaggacaca gccacatatt actgtgcaag aggatcttat 300

tactatagcg cgtcgggcta ctttgattac tggggccacg gagtcatggt cacagtctcc 360

tca 363

<210> 179

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-L1

<400> 179

ctagcaagtg aggacattta cagtgattta gca 33

<210> 180

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-L2

<400> 180

aatgcaaata acttgcaaaa t 21

<210> 181

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-L3

<400> 181

caacaatata acagttatcc gaacacg 27

<210> 182

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-H1

<400> 182

acaacttact ggggc 15

<210> 183

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-H2

<400> 183

cacataagta acagtggtag tactaattac aacccatctc tcaaaagt 48

<210> 184

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-H3

<400> 184

ggatcttatt actatagcgc gtcgggctac tttgattac 39

<210> 185

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-VL

<400> 185

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

1 5 10 15

Glu Thr Val Asn Ile Gly Cys Leu Ala Ser Glu Asp Ile Tyr Ser Asp

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 186

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-VH

<400> 186

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

1 5 10 15

Ser Leu Ser Leu Ile Cys Ser Val Thr Gly Tyr Ser Ile Thr Thr Thr

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Ile

35 40 45

Gly His Ile Ser Asn Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gly Ser Tyr Tyr Tyr Ser Ala Ser Gly Tyr Phe Asp Tyr Trp Gly

100 105 110

His Gly Val Met Val Thr Val Ser Ser

115 120

<210> 187

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-L1

<400> 187

Leu Ala Ser Glu Asp Ile Tyr Ser Asp Leu Ala

1 5 10

<210> 188

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-L2

<400> 188

Asn Ala Asn Asn Leu Gln Asn

1 5

<210> 189

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-L3

<400> 189

Gln Gln Tyr Asn Ser Tyr Pro Asn Thr

1 5

<210> 190

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-H1

<400> 190

Thr Thr Tyr Trp Gly

1 5

<210> 191

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-H2

<400> 191

His Ile Ser Asn Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 192

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 45H1-CDR-H3

<400> 192

Gly Ser Tyr Tyr Tyr Ser Ala Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 193

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 14E11-VL (control)

<400> 193

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Leu Thr Ser Tyr Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

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

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Lys Thr Pro Tyr

85 90 95

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

100 105

<210> 194

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 14E11-VH (control)

<400> 194

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

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

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

35 40 45

Gly Met Ile Trp Gly Asp Gly Arg Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Tyr Gly Ser Lys Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser

115

<210> 195

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 1A6-VL (control)

<400> 195

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

1 5 10 15

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

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 196

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: 1A6-VH (control)

<400> 196

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Thr Ser Val Asp Ala Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Lys Arg Ser Ser Val Val Ala His Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

115 120

<210> 197

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-19F6-VL

<400> 197

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

1 5 10 15

Arg Val Thr Ile Thr Cys Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Asn Val

35 40 45

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

50 55 60

Gly Ser Ile Asp Gly Ser Gly Asn Ser Ala Ser Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Ala Glu Asp Ala Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Ser

85 90 95

Asn Ile Asp Phe Asn Pro Val Phe Gly Gly Gly Thr Lys Leu Glu Val

100 105 110

Lys

<210> 198

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-19F6-VH

<400> 198

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg His Pro Phe Asn Asn Phe Gly Ile Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 199

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-34F8-VL

<400> 199

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

1 5 10 15

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

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Val Leu Leu Tyr Gly Val Ser Asn Arg Leu Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

Thr His Gly Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr

<210> 200

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-34F8-VH

<400> 200

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

His Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Met Trp Arg Asp Gly Asp Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Gly Gly Thr Leu Thr Thr Pro Phe Thr Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 201

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-42A5-VL

<400> 201

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

1 5 10 15

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

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

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

100 105 110

Arg Thr

<210> 202

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-42A5-VH

<400> 202

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

His Leu His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Met

35 40 45

Gly Leu Met Trp Arg Asp Gly Asp Thr Ser Tyr Asn Ser Arg Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Gly Met Thr Leu Ala Thr Pro Phe Leu Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 203

<211> 625

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> amino acid sequence of human FXI

<400> 203

Met Ile Phe Leu Tyr Gln Val Val His Phe Ile Leu Phe Thr Ser Val

1 5 10 15

Ser Gly Glu Cys Val Thr Gln Leu Leu Lys Asp Thr Cys Phe Glu Gly

20 25 30

Gly Asp Ile Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gln Val

35 40 45

Val Cys Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu

50 55 60

Ser Pro Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp

65 70 75 80

Ser Val Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala Ile Ser

85 90 95

Gly Tyr Ser Phe Lys Gln Cys Ser His Gln Ile Ser Ala Cys Asn Lys

100 105 110

Asp Ile Tyr Val Asp Leu Asp Met Lys Gly Ile Asn Tyr Asn Ser Ser

115 120 125

Val Ala Lys Ser Ala Gln Glu Cys Gln Glu Arg Cys Thr Asp Asp Val

130 135 140

His Cys His Phe Phe Thr Tyr Ala Thr Arg Gln Phe Pro Ser Leu Glu

145 150 155 160

His Arg Asn Ile Cys Leu Leu Lys His Thr Gln Thr Gly Thr Pro Thr

165 170 175

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

180 185 190

Cys Ala Leu Ser Asn Leu Ala Cys Ile Arg Asp Ile Phe Pro Asn Thr

195 200 205

Val Phe Ala Asp Ser Asn Ile Asp Ser Val Met Ala Pro Asp Ala Phe

210 215 220

Val Cys Gly Arg Ile Cys Thr His His Pro Gly Cys Leu Phe Phe Thr

225 230 235 240

Phe Phe Ser Gln Glu Trp Pro Lys Glu Ser Gln Arg Asn Leu Cys Leu

245 250 255

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

260 265 270

Lys Ala Leu Ser Gly Phe Ser Leu Gln Ser Cys Arg His Ser Ile Pro

275 280 285

Val Phe Cys His Ser Ser Phe Tyr His Asp Thr Asp Phe Leu Gly Glu

290 295 300

Glu Leu Asp Ile Val Ala Ala Lys Ser His Glu Ala Cys Gln Lys Leu

305 310 315 320

Cys Thr Asn Ala Val Arg Cys Gln Phe Phe Thr Tyr Thr Pro Ala Gln

325 330 335

Ala Ser Cys Asn Glu Gly Lys Gly Lys Cys Tyr Leu Lys Leu Ser Ser

340 345 350

Asn Gly Ser Pro Thr Lys Ile Leu His Gly Arg Gly Gly Ile Ser Gly

355 360 365

Tyr Thr Leu Arg Leu Cys Lys Met Asp Asn Glu Cys Thr Thr Lys Ile

370 375 380

Lys Pro Arg Ile Val Gly Gly Thr Ala Ser Val Arg Gly Glu Trp Pro

385 390 395 400

Trp Gln Val Thr Leu His Thr Thr Ser Pro Thr Gln Arg His Leu Cys

405 410 415

Gly Gly Ser Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala Ala His Cys

420 425 430

Phe Tyr Gly Val Glu Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile

435 440 445

Leu Asn Gln Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln

450 455 460

Glu Ile Ile Ile His Asp Gln Tyr Lys Met Ala Glu Ser Gly Tyr Asp

465 470 475 480

Ile Ala Leu Leu Lys Leu Glu Thr Thr Val Asn Tyr Thr Asp Ser Gln

485 490 495

Arg Pro Ile Cys Leu Pro Ser Lys Gly Asp Arg Asn Val Ile Tyr Thr

500 505 510

Asp Cys Trp Val Thr Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys Ile

515 520 525

Gln Asn Thr Leu Gln Lys Ala Lys Ile Pro Leu Val Thr Asn Glu Glu

530 535 540

Cys Gln Lys Arg Tyr Arg Gly His Lys Ile Thr His Lys Met Ile Cys

545 550 555 560

Ala Gly Tyr Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly

565 570 575

Gly Pro Leu Ser Cys Lys His Asn Glu Val Trp His Leu Val Gly Ile

580 585 590

Thr Ser Trp Gly Glu Gly Cys Ala Gln Arg Glu Arg Pro Gly Val Tyr

595 600 605

Thr Asn Val Val Glu Tyr Val Asp Trp Ile Leu Glu Lys Thr Gln Ala

610 615 620

Val

625

<210> 204

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-42A5-VL

<400> 204

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

1 5 10 15

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

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

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

100 105 110

Arg Thr

<210> 205

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-42A5-VH

<400> 205

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

Ala Glu Ile Trp Arg Asp Gly Asp Thr Ser Tyr Asn Ser Arg Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Gly Met Thr Leu Ala Thr Pro Phe Leu Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 206

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-34F8-VL

<400> 206

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

1 5 10 15

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

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Val Leu Leu Tyr Gly Val Ser Asn Arg Leu Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

Thr His Gly Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr

<210> 207

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-34F8-VH

<400> 207

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Thr Tyr

20 25 30

His Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Met Trp Arg Asp Gly Asp Thr Tyr Tyr Asn Pro Lys Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Gly Gly Thr Leu Thr Thr Pro Phe Thr Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 208

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-19F6-VL

<400> 208

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

1 5 10 15

Arg Val Thr Ile Thr Cys Glu Arg Ser Ser Gly Asp Ile Gly Asp Ser

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Asn Val

35 40 45

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

50 55 60

Gly Ser Ile Asp Gly Ser Gly Asn Ser Ala Ser Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Ala Glu Asp Ala Ala Asp Tyr Phe Cys Gln Ser Tyr Asp Ser

85 90 95

Asn Ile Asp Phe Asn Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val

100 105 110

Leu

<210> 209

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic: h-19F6-VH

<400> 209

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg His Pro Phe Asn Asn Phe Gly Ile Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser

115 120

Claims

1. An isolated anti-FXI or anti-FXIa antibody that specifically binds human FXI or FXIa, wherein the antibody comprises an amino acid sequence selected from SEQ ID NOs: 123-128, sequences having at least 90% identity, or immunologically active portions thereof.

2. The antibody of claim 1, wherein said antibody specifically binds the a3 domain of human FXI or FXIa.

3. The antibody of claim 1, wherein the antibody comprises a pair of sequences selected from the following pairs of sequences: SEQ ID NO: 121-122, SEQ ID NO: 199-200, and SEQ ID NO: 206, 207, and sequences having at least 90% identity.

4. A pharmaceutical composition comprising the antibody of any one of claims 1-3.

5. Use of the antibody of any one of claims 1-3 in the manufacture of a medicament for inhibiting blood clot formation in a subject.

6. Use of the pharmaceutical composition of claim 4 in the manufacture of a medicament for inhibiting blood clot formation in a subject.

7. Use of an antibody of any one of claims 1-3 in the manufacture of a medicament for treating or preventing thrombosis or a complication or disorder associated with thrombosis, wherein the amount of the antibody does not impair hemostatic function of the subject.

8. Use of the pharmaceutical composition of claim 4 in the manufacture of a medicament for treating or preventing thrombosis or a complication or disorder associated with thrombosis, wherein the amount of the composition does not impair hemostatic function of the subject.

9. Use of an antibody of any one of claims 1-3 in the manufacture of a medicament for treating or preventing sepsis, wherein the amount of the antibody does not impair hemostatic function of the subject.

10. Use of the pharmaceutical composition of claim 4 in the manufacture of a medicament for treating or preventing sepsis, wherein the amount of the composition does not impair hemostatic function of the subject.

11. A method of making the antibody of any one of claims 1-3, comprising expressing in a host cell a nucleic acid encoding the antibody cloned in an expression vector.

12. The method of claim 11, further comprising purifying the expressed antibody from the host cell.

13. The method of claim 11, wherein the expression vector is a pTT5 vector or a pcDNA3 vector.

14. The method of claim 11, wherein the host cell is a CHO cell or a HEK193T cell.

15. An antibody or functional fragment thereof, or an immunologically active portion thereof, prepared by the method of any one of claims 11-14.

16. The antibody of claim 15, wherein the antibody has been post-translationally modified.