HK1229230B

HK1229230B - Igf-1r antibody-drug-conjugate and its use for the treatment of cancer

Info

Publication number: HK1229230B
Application number: HK17102923.9A
Authority: HK
Inventors: I．里拉特; M.佩雷斯; L．格奇; M．布鲁萨斯; C．博-拉沃尔; J-F．哈尔威; T．钱皮恩; A.罗伯特
Original assignee: 皮埃尔法布雷医药公司
Priority date: 2014-04-25
Filing date: 2015-04-27
Publication date: 2019-06-14

Description

IGF-1R antibody-drug-conjugates and uses thereof for the treatment of cancer

Technical Field

The present invention relates to an antibody-drug-conjugate capable of binding to IGF-1R. In one aspect, the invention relates to an antibody-drug-conjugate comprising an antibody capable of binding to IGF-1R conjugated to at least one drug selected from dolastatin 10(dolastatin 10) and derivatives of auristatin (auristatin). The invention also includes methods of treatment, and uses of the antibody-drug-conjugates for the treatment of cancer.

Background

the insulin-like growth factor 1 receptor, known as IGF-1R (or sometimes referred to as IGF1R or IGF-IR), is a receptor with tyrosine kinase activity that has 70% homology to insulin receptor IR IGF-1R is a glycoprotein with a molecular weight of about 350,000, a heterotetrameric (heterameric) receptor in which each half of the linkage via a disulfide bridge is composed of an extracellular α -subunit and a transmembrane β -subunit IGF-1R binds IGF1 and IGF2 with very high affinity (Kd #1nM), but is equally capable of binding to insulin with 100 to 1000 fold less affinity, in contrast, IR binds to insulin with very high affinity, although IGF binds to insulin receptor with only 100 fold less affinity, IGF-1R and the tyrosine kinase domain of IR have high sequence homology, although the weaker segments are involved in the terminal part region of the α -subunit and the C-terminal part of the β -subunit, are in the α -subunit, and the differential signaling of the opposite mitotic pathway leading to differential activation of IGF-1 and β -subunit pathways, respectively, and thus the differential signaling of the activation of IGF-1 and the opposite mitogenic pathways.

Cytoplasmic tyrosine kinase protein is activated by binding of a ligand to the extracellular domain of the receptor. Activation of kinases in turn involves stimulation of different intracellular substrates including IRS-1, IRS-2, Shc and Grb 10. The two major substrates of IGF-1R are IRS and Shc, which mediate most of the growth and differentiation effects associated with the linkage of IGF to this receptor through the activation of a number of downstream effectors. Thus, the availability of the substrate may determine the final biological effect associated with the activation of IGF-1R. When IRS-1 predominates, the cells tend to proliferate and transform. When Shc predominates, cells tend to differentiate. The pathway that appears to be primarily involved in protection against the effects of apoptosis is the phosphatidyl-inositol 3-kinase (PI 3-kinase) pathway.

The role of the IGF system in carcinogenesis (carcinogenesis) has been the subject of intensive research in the last decade. This interest follows from the discovery that, in addition to its mitogenic and anti-apoptotic properties, IGF-1R appears to be essential for the establishment and maintenance of the transformed phenotype. In fact, it has been well established that, in many cell types, overexpression or constitutive activation of IGF-1R leads to cell growth that is independent of support in media lacking fetal bovine serum and leads to tumor formation in nude mice. This is not a unique property in itself, as the many products of an overexpressed gene can transform cells, including a large number of growth factor receptors. However, the key finding that it has clearly been demonstrated that IGF-1R plays a major role in transformation has demonstrated that IGR-1R-cells (in which the gene encoding IGF-1R has been inactivated) are completely difficult to transform by different agents that are normally capable of transforming cells, such as the E5 protein of bovine papilloma virus, overexpression of EGFR or PDGFR, the T antigen of SV40, activated ras or a combination of the last two factors.

IGF-1R is expressed in a wide variety of tumors and tumor lines, and IGF amplifies tumor growth via their linkage to IGF-1R. Other arguments that support the role of IGF-1R in carcinogenesis come from studies using murine monoclonal antibodies directed against the receptor, or using a dominant negative mutant of IGF-1R (negative dominant). Indeed, murine monoclonal antibodies directed against IGF-1R inhibit the proliferation of various cell lines in culture and inhibit the growth of tumor cells in vivo. Likewise, dominant negative mutants of IGF-1R have been shown to inhibit tumor proliferation.

A number of projects have begun to develop naked IGF-1R antibodies for the treatment of cancer. However, to date, none of these projects have been successful and there are no anti-IGF-1R antibodies on the market.

Furthermore, a series of clinical trials involving anti-IGF-1R antibodies combined with anti-EGFR antibodies to target EGFR and IGF-1R have failed, as none of these antibodies are able to treat KRAS mutant patients.

Therefore, IGF-1R is not now considered to be a major target, and IGF-1R is no longer considered to be of particular interest in the study of potential therapeutic antibodies.

However, it must also be noted that efforts to generate IGF-1R antibodies have focused on naked antibodies, i.e., antibodies that are useful by their intrinsic properties. In this sense, IGF-1R is considered to be a target that is not suitable for the production of ADCs such as antibody-drug-conjugates (referred to as "ADCs"), since IGF-1R is described as a target that is also widely expressed by normal cells (including blood vessels). In this sense, it can be noted that the most recent IGF-1R antibody (i.e., AVE1642) was developed as a naked antibody without drug. It is identical to other IGF-1R antibodies currently in development and to all those that failed in clinical trials.

In this context, the present invention relates to an ADC or conjugate, and its use for the treatment of cancer, more particularly cancers expressing IGF-1R.

ADCs combine the binding specificity of an antibody with the potency of a drug (e.g., a cytotoxic agent). In recent years, technologies related to the development of monoclonal antibodies, the use of more effective drugs, and the design of chemical linkers covalently binding these components have rapidly progressed.

The use of ADCs allows for the local delivery of drugs that, if administered as unconjugated drugs, may produce unacceptable levels of toxicity to normal cells.

In other words, the maximum efficacy with the least toxicity is therefore sought. Efforts to design and improve ADCs have focused on antibody selectivity as well as drug mechanism of action, drug attachment, drug/antibody ratio (loading or DAR) and drug release properties. The drug moiety may exert its cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, impairment of proteasome, ribosomal function, protein synthesis and/or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated with large antibodies.

Each antibody must be characterized individually, appropriate linkers designed, and appropriate cytotoxic agents identified that retain their potency after delivery to tumor cells. The density of antigen at the cancer target must be considered, as well as whether normal tissue expresses the target antigen. Other considerations include whether the entire ADC is internalized upon binding to the target; whether cytostatic or cytotoxic drugs are preferred when considering possible normal tissue exposure and/or the type and stage of cancer being treated; and whether the linker connecting the antibody to the drug payload is cleavable or non-cleavable. Furthermore, for future development processes of compounds, the conjugation ratio of antibody to drug moiety must be sufficient without compromising the binding activity of the antibody and/or the potency of the drug, and without altering the physicochemical properties of the ADC leading to aggregation or deleterious properties.

ADCs are complex biological molecules and the challenge of developing effective ADCs remains a significant problem.

Disclosure of Invention

Summary of The Invention

The present invention aims to solve this problem and relates to an ADC of the following formula (I):

Ab-(L-D)_n(I)

or a pharmaceutically acceptable salt thereof,

wherein

Ab is an antibody or antigen-binding fragment thereof capable of binding to human IGF-1R, said Ab selected from the group consisting of:

i) an antibody comprising three heavy chain CDRs of sequences SEQ ID nos.1, 2 and 3 and three light chain CDRs of sequences SEQ ID nos. 4, 5 and 6;

ii) an antibody that competes with the antibody of i) for binding to IGF-1R; and

iii) an antibody that binds to the same epitope of IGF-1R as the antibody of i);

l is a linker;

d is a drug moiety of the following formula (II):

wherein:

R₂is COOH, COOCH₃Or a thiazolyl group;

R₃is H or (C)₁-C₆) An alkyl group;

R₉is H or (C)₁-C₆) An alkyl group;

m is an integer between 1 and 8;

the wavy line indicates the point of connection with L; and

n is 1 to 12.

One embodiment of the invention relates to an ADC wherein Ab is selected from:

a) an antibody comprising three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11;

b) an antibody comprising three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3 and three light chain CDRs of sequences SEQ ID nos. 10, 5 and 11;

c) an antibody comprising three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 12; and

d) an antibody comprising three heavy chain CDRs of sequences SEQ ID nos. 8, 2 and 3 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11.

One embodiment of the invention relates to an ADC wherein Ab is selected from:

a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.13 and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;

b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.14 and three light chain CDRs of sequences SEQ ID Nos. 10, 5 and 11;

c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.15 and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12;

d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.16 and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and

e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.17 and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12.

One embodiment of the invention relates to an ADC wherein Ab is selected from:

a) an antibody comprising a light chain variable domain of sequence SEQ ID No.18 and three heavy chain CDRs of sequences SEQ ID Nos. 7,2 and 3;

b) an antibody comprising a light chain variable domain of sequence SEQ ID No.19 and three heavy chain CDRs of sequences SEQ ID Nos. 7,2 and 3;

c) an antibody comprising a light chain variable domain of sequence SEQ ID No.20 and three heavy chain CDRs of sequences SEQ ID Nos. 7,2 and 3;

d) an antibody comprising a light chain variable domain of sequence SEQ ID No.21 and three heavy chain CDRs of sequences SEQ ID Nos. 8, 2 and 3; and

e) an antibody comprising a light chain variable domain of sequence SEQ ID No.22 and three heavy chain CDRs of sequences SEQ ID Nos. 7,2 and 3.

In one embodiment, the invention relates to an ADC wherein Ab is selected from:

i) antibodies 208F2, 212a11, 214F8, 219D6, and 213B 10;

iii) an antibody that binds to the same epitope of IGF-1R as the antibody of i).

One embodiment of the invention relates to an ADC wherein Ab is a humanized antibody.

One embodiment of the invention relates to an ADC wherein Ab is selected from the group consisting of antibodies comprising:

a) heavy chains of CDR-H1, CDR-H2 and CDR-H3 having the sequences SEQ ID nos. 7,2 and 3, respectively, and FR1, FR2 and FR3 derived from the human germline IGHV1-46 × 01(SEQ ID No.46), and FR4 derived from the human germline IGHJ4 × 01(SEQ ID No. 48); and

b) light chains of CDR-L1, CDR-L2 and CDR-L3 having the sequences SEQ ID Nos. 9, 5 and 11, respectively, and FR1, FR2 and FR3 derived from the human germline IGKV1-39 × 01(SEQ ID No.47), and FR4 derived from the human germline IGKJ4 × 01(SEQ ID No. 49).

In one embodiment of the invention, Ab is selected from:

a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.33 or any sequence showing at least 80% identity to SEQ ID No.33 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11; and

b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.34 or any sequence showing at least 80% identity to SEQ ID No.34 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11.

In one embodiment of the invention, Ab is selected from:

a) an antibody comprising a light chain variable domain of sequence SEQ ID No.35 or any sequence showing at least 80% identity to SEQ ID No.35 and three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3; and

b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.36 or any sequence showing at least 80% identity to SEQ ID No.36 and three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3.

In one embodiment of the invention, Ab is selected from:

a) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.37 or any sequence showing at least 80% identity to SEQ ID No.37 and a light chain of sequence SEQ ID No.39 or any sequence showing at least 80% identity to SEQ ID No. 39; and

b) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.38 or any sequence showing at least 80% identity to SEQ ID No.38 and a light chain of sequence SEQ ID No.40 or any sequence showing at least 80% identity to SEQ ID No. 40.

In one embodiment of the invention, Ab is selected from:

a) a heavy chain variable domain comprising a sequence selected from SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence having at least 80% identity to SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80; and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11;

b) a light chain variable domain comprising a sequence selected from SEQ ID nos. 57 and 60 or any sequence having at least 80% identity to SEQ ID nos. 57 or 60; and three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3; and

c) a heavy chain variable domain comprising a sequence selected from SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence having at least 80% identity to SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80; and a light chain variable domain selected from the sequence of SEQ ID No.57 or 60 or any sequence having at least 80% identity to SEQ ID No.57 or 60.

In one embodiment of the invention, Ab is selected from:

a) a heavy chain of a sequence selected from SEQ ID nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 and 81 or any sequence having at least 80% identity to SEQ ID nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 or 81; and

b) a light chain selected from the sequences of SEQ ID Nos. 59 and 61 or any sequence having at least 80% identity to SEQ ID Nos. 59 or 61.

One embodiment of the present invention relates to an ADC wherein L is a linker of formula (III):

wherein

L₂Is (C)₄-C₁₀) Cycloalkyl-carbonyl group, (C)₂-C₆) Alkyl or (C)₂-C₆) Alkyl-carbonyl;

w is an amino acid unit; w is an integer between 0 and 5;

y is PAB-carbonyl, wherein PAB isy is 0 or 1;

asterisks indicate points of attachment to D; and

the wavy line indicates the point of attachment to Ab.

One embodiment of the present invention relates to an ADC, wherein L₂Is of the formula:

wherein

Star sign of AND (W)_wA point of connection; and

the wavy line represents the point of attachment to the nitrogen atom of the maleimide moiety of the formula:

in one embodiment of the invention, W ═ 0, or W ═ 2 then (W)_wSelected from:

wherein

Asterisk denotes the sum (Y)_yA point of connection; and

wavy line representation and L₂The point of connection.

One embodiment of the invention relates to an ADC, wherein L is selected from:

where the asterisk indicates the point of attachment to D and the wavy line indicates the point of attachment to Ab.

One embodiment of the present invention relates to an ADC, wherein (L-D) is selected from:

where the wavy line indicates the point of attachment to the Ab.

One embodiment of the invention relates to an ADC having a formula selected from:

and a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable salts thereof,

wherein Ab is selected from:

i) antibodies 208F2, 212a11, 214F8, 219D6, and 213B 10;

One embodiment of the invention relates to an ADC wherein n is 2.

One embodiment of the invention relates to an ADC wherein n is 4.

One embodiment of the invention relates to an ADC, for use as a medicament.

One embodiment of the invention relates to a composition comprising an ADC as described above.

One embodiment of the present invention relates to a composition further comprising a pharmaceutically acceptable carrier (vehicle).

One embodiment of the invention relates to a composition for use in the treatment of cancer expressing IGF-1R or IGF-1R associated cancer.

Cancers that express IGF-1R or IGF-1R-associated cancers include tumor cells that express or overexpress all or a portion of IGF-1R on their surface.

One embodiment of the invention relates to a composition wherein said cancer expressing IGF-1R is selected from the group consisting of breast cancer, colon cancer, esophageal cancer, hepatocellular cancer, gastric cancer, glioma, lung cancer, melanoma, osteosarcoma, ovarian cancer, prostate cancer, rhabdomyosarcoma, renal cancer, thyroid cancer, endometrial cancer, mesothelioma, oral squamous cell carcinoma, and any drug-resistant cancer.

One embodiment of the present invention relates to a method for treating an IGF-1R-expressing cancer in a subject in need thereof, comprising administering to the subject an effective amount of at least one antibody-drug-conjugate or a composition according to the present invention.

One embodiment of the present invention relates to a kit comprising at least i) an antibody-drug-conjugate and/or a composition as described above and ii) a syringe or vial or ampoule into which the antibody-drug-conjugate and/or composition is placed.

Detailed Description

I-antibody (Ab)

The terms "antibody (antibody)", "antibodies (antibodies)", "Ab", "MAb", or "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies, isolated, engineered or recombinant antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies or multispecific antibodies (e.g., bispecific antibodies) and antibody fragments thereof, so long as they exhibit the desired biological activity.

In one embodiment, the antibody of the ADC of the invention consists of a recombinant antibody. The term "recombinant antibody" refers to an antibody produced by expression of recombinant DNA in a living cell. By using the genetic recombination laboratory method well known to those skilled in the art, DNA sequences that are not found in biological organisms are generated to obtain recombinant antibodies to the ADCs of the present invention.

In another embodiment, the antibody of the ADC of the invention consists of a chemically synthesized antibody.

More particularly, the molecule consists of a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, namely CL. The VH and VL regions may be further subdivided into regions of hypervariability, known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

An "antigen-binding fragment" or "IGF-1R-binding fragment" of an antibody of an ADC according to the invention is intended to mean any peptide, polypeptide or protein that retains the ability to bind to the target (also commonly referred to as antigen) of the antibody.

In one embodiment, the "antigen-binding fragment" is selected from the group consisting of Fv, scFv (sc for single chain), Fab, F (ab')₂Fab ', scFv-Fc fragment or diabody, or any fragment whose half-life has been extended by chemical modification such as the addition of a polyalkylene glycol such as polyethylene glycol ("PEGylation") (referred to as Fv-PEG, scFv-PEG, Fab-PEG, F (ab')₂-pegylated fragments of PEG or Fab' -PEG) ("PEG" means polyethylene glycol) having at least one of the characteristic CDRs of an antibody according to the invention. Preferably, said "antigen-binding fragments" will comprise or will consist of a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being sufficient to retain the same binding specificity as the antibody from which it originates, and sufficient affinity for the target, preferably at least equal to 1/100, more preferably at least 1/10, of the affinity of the antibody from which it originates. More preferably, said "antigen-binding fragment" will comprise or will consist of at least the three CDRs of the heavy variable chain (CDR-H1, CDR-H2 and CDR-H3) and the three CDRs of the light variable chain (CDR-L1, CDR-L2 and CDR-L3) of the antibody from which they are derived.

"binding", and the like, refer to an antibody, or any antigen-binding fragment thereof, forming a complex with an antigen that is relatively stable under physiological conditions. Specific binding is characterized by at least about 1x10^-6Equilibrium dissociation constant of M. Methods for determining whether two molecules bind are well known to those skilled in the art and include, for example, equilibrium dialysis, surface plasmon resonance, radiolabelling analysis, and the like. For the avoidance of doubt, this does not mean that the antibody is not capable of binding to or interfering with another antigen at low levels. However,as one embodiment, the antibody binds only to the antigen.

As used in this specification, the expression "IGF-1R antibody" should be construed to be similar to "anti-IGF-1R antibody" and refers to antibodies that are capable of binding to IGF-1R.

In one embodiment of the present application, the epitope of the antibody is preferably localized to the extracellular domain of human IGF-1R (also known as IGF-1R ECD).

In a particular embodiment, the antibody or any antigen binding fragment thereof can be at 10x10^-10To 1x10^-10More preferably 8x10^-10To 2x10^-10EC of (1)₅₀Binds to IGF-1R.

The term half maximal Effective Concentration (EC)₅₀) Corresponding to the concentration of drug, antibody or poison that induces a response intermediate between the baseline and maximum values after some specified exposure time. It is commonly used as a measure of drug efficacy. Thus, EC of the fractionated dose response curve₅₀Represents the concentration of the compound at which 50% of the maximum effect of the compound is observed. Quantification of EC of dose response Curve₅₀Represents the concentration of the compound at which 50% of the population exhibits a response after a particular duration of exposure. Concentration measurements typically follow a sigmoidal curve, increasing rapidly with relatively small concentration changes. This can be determined mathematically by derivation of a best fit line.

As a preferred embodiment, the EC determined in the present invention₅₀The efficacy of the antibodies in binding to IGF-1R ECD exposed on human tumor cells was characterized. Determination of EC Using FACS analysis₅₀And (4) parameters. EC (EC)₅₀The parameter reflects the antibody concentration at which 50% of the maximum binding on human IGF-1R expressed on human tumor cells is obtained. Each EC was calculated as the midpoint of the dose response curve using a four parameter regression curve fitting program (Prism Software)₅₀The value is obtained. This parameter has been selected as representative of the physiological/pathological condition.

The term "epitope" is the region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and those residues that have an affinity that directly favors interaction. Epitopes may also be conformational, i.e. consisting of nonlinear amino acids. In certain embodiments, an epitope may include a determinant (determinant), which is a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and may, in certain embodiments, have particular three-dimensional structural characteristics, and/or particular charge characteristics.

Competition for IGF-1R binding can be determined by any method or technique known to those skilled in the art, such as, but not limited to, radiation, surface plasmon resonance (Biacore), ELISA, flow cytometry, and the like. "competing for binding to IGF-1R" means at least 20%, preferably at least 50%, more preferably at least 70% competition.

Determination of binding to the same epitope can be determined by any method or technique known to those skilled in the art, such as, but not limited to, radiation, surface plasmon resonance (Biacore), ELISA, flow cytometry, and the like. "binding to the same epitope of IGF-1R" refers to competition of at least 20%, preferably at least 50%, more preferably at least 70%.

As mentioned above, contrary to general knowledge, the present invention focuses on specific IGF-1R antibodies that exhibit high internalization capacity upon IGF-1R binding. As used herein, an "internalized" or "internalized" (both expressions are similar) antibody is an antibody that is taken up by (meaning "taken into") a mammalian cell upon binding to IGF-1R on the cell. The antibody is of interest as part of an ADC, and therefore it localizes or directs the attached cytotoxin to the targeted cancer cell. Once internalized, the cytotoxin triggers cancer cell death.

Surprisingly, the antibody according to the invention shows the same sequence for all of CDR-H2, CDR-H3 and CDR-L2, the other 3 CDRs being different. This observation appears to be clear (coherent) in that it is part of the common general knowledge about the binding specificity of antibodies, CDR-H3 being described as being the most important and relevant for the recognition of epitopes.

The key to the success of ADC therapy is believed to be the target antigen specificity and internalization of the antigen-antibody complex into the cancer cell. Clearly, non-internalizing antigen delivers cytotoxic agents less efficiently than internalizing antigens. The internalisation process is variable between antigens and depends on a variety of parameters which can be influenced by the antibody.

In ADCs, the cytotoxic agent confers cytotoxic activity, the antibody used is responsible for specificity against cancer cells, and the carrier (vector) is responsible for entry into the cell to exert the cytotoxic agent properly. Thus, to improve ADCs, antibodies may exhibit a high capacity for internalization into targeted cancer cells. The efficiency of antibody-mediated internalization varies significantly depending on the epitope targeted. The selection of an effective internalizing IGF-1R antibody requires various experimental data that not only studies IGF-1R down-regulation, but also studies the internalization of IGF-1R antibody into cells.

In one embodiment, internalization of antibodies of ADCs according to the present invention may be assessed by immunofluorescence or FACS (flow cytometry) as exemplified herein below, or any method or process known to those of skill in the art, particularly for internalization mechanisms. In a preferred embodiment, the antibody of the ADC according to the invention may induce internalization of at least 30%, preferably 50%, more preferably 80%, upon binding to IGF-1R.

Upon binding of the antibody to the ECD of the IGF-1R, the IGF-1R/antibody complex is internalized and induces a decrease in the amount of IGF-1R on the cell surface. This reduction can be quantified by any method known to those skilled in the art, such as, by way of non-limiting example, western-blot, FACS and immunofluorescence.

In one embodiment, this reduction may preferably be measured by FACS (thereby reflecting internalization) and expressed as the difference or Δ between the Mean Fluorescence Intensity (MFI) measured at 4 ℃ and the MFI measured at 37 ℃ after 4 hours of incubation with the antibody.

As a non-limiting example, this delta was determined based on the MFI obtained with untreated cells and with antibody-treated cells using i) breast cancer cells MCF7 after 4 hours of incubation with the antibodies described herein, and ii) a second antibody labeled with Alexa 488. This parameter is defined as being calculated using the following equation: delta (MFI)_4℃–MFI_37℃)。

This difference between MFIs reflects IGF-1R down-regulation, as MFI is proportional to IGF-1R expressed on the cell surface.

In an advantageous aspect, the antibody is triggered by a delta (MFI) of at least 280, preferably at least 400, in MCF-7_4℃–MFI_37℃) The antibody of (1).

In more detail, the Δ mentioned above can be measured according to the following method, which must be considered as an illustrative and non-limiting example:

a) treating and incubating tumor cells of interest with the antibodies of the invention in cold (4 ℃) or warm (37 ℃) complete culture medium;

b) treating the treated cells and untreated cells of step a) with a second antibody in parallel;

c) measuring the MFI (representing the amount of IGF-1R present on the surface) of the cells treated and untreated with a second labeled antibody capable of binding to the antibody of the invention; and

d) the MFI obtained with untreated cells was subtracted from the MFI obtained with treated cells to calculate Δ.

From this Δ MFI, percent internalization can be determined as:

100x(MFI_4℃-MFI_37℃)/MFI_4℃.

in MCF7, the antibody of the ADC according to the invention preferably shows a percent internalization of 50% to 99%, 70% to 90%, preferably 75% to 87%.

The particular advantage of the antibodies described herein depends on their internalization rate.

It is generally known that for ADCs, it is desirable that the antibody used exhibits a fast internalization rate, preferably within 24 hours, more preferably within 12 hours, even more preferably within 6 hours of administration of the antibody.

In the present invention, the rate of internalization (also referred to as cell surface bound antibody reduction or cell surface antibody decay) is expressed as t1/2 (half-life) and corresponds to the time required to obtain a 50% reduction in Δ MFI (this aspect will be clearly understood from the examples below).

A particular advantage is that the antibody of the ADC of the invention has a t1/2 of 5 to 25 minutes, preferably 10 to 20 minutes.

A particular embodiment of the invention relates to an ADC in which antibody Ab comprises three heavy chain CDRs having the CDR-H2 of sequence SEQ ID No.2 and the CDR-H3 of sequence SEQ ID No.3, and three light chain CDRs having the CDR-L2 of sequence SEQ ID No. 5.

A particular embodiment of the invention relates to an ADC in which antibody Ab comprises three heavy chain CDRs of sequences SEQ ID nos.1, 2 and 3 and three light chain CDRs of sequences SEQ ID nos. 4, 5 and 6.

One embodiment of the ADC comprises an antibody comprising three heavy chain CDRs comprising or consisting of the sequences SEQ ID nos.1, 2 and 3 or any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID nos.1, 2 or 3 and three light chain CDRs comprising or consisting of the sequences SEQ ID nos. 4, 5 and 6 or any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID nos. 4, 5 or 6.

In another embodiment, the antibody, or any antigen-binding fragment thereof, comprises three heavy chain CDRs comprising the sequences SEQ ID nos.1, 2, and 3; the three light chain CDRs comprise the sequences SEQ ID Nos. 4, 5 and 6.

Unique numbering of IMGTs has been defined to compare variable domains regardless of antigen receptor, chain type or species [ Lefranc m. -p, Immunology Today 18,509(1997)/Lefranc m. -p ], The Immunology, 7,132-136(1999)/Lefranc, m. -p, Pommi e, c, Ruiz, m. In the unique numbering of IMGT, conserved amino acids always have the same position, e.g., cysteine 23 (CYS 1), tryptophan 41 (conserved-TRP), hydrophobic amino acid 89, cysteine 104 (CYS 2), phenylalanine or tryptophan 118(J-PHE or J-TRP). The unique numbering of the IMGT provides standardized delineation of framework regions (FR 1-IMGT: positions 1 to 26, FR 2-IMGT: 39 to 55, FR 3-IMGT: 66 to 104 and FR 4-IMGT: 118 to 128), and standardized delineation of complementarity determining regions (CDR 1-IMGT: 27 to 38, CDR 2-IMGT: 56 to 65 and CDR 3-IMGT: 105 to 117). Since the gaps represent unoccupied positions, the CDR-IMGT lengths (shown in parentheses and separated by dots, e.g., [8.8.13]) become critical information. In the 2D illustration called IMGT colliers trees [ Ruiz, m. and Lefranc, m. -p., Immunogenetics,53,857-883(2002)/Kaas, q. and Lefranc, m. -p., Current Bioinformatics,2,21-30(2007) ], and the 3D structure of IMGT/3D structure-DB [ Kaas, q., Ruiz, m. and Lefranc, m. -p., T cell extractor and MHC structure.

It must be understood that, without contradictory statements in this specification, complementarity determining regions or CDRs refer to the hypervariable regions of the heavy and light chains of an immunoglobulin as defined according to the IMGT numbering system.

However, the CDRs may also be defined according to the Kabat numbering system (Kabat et al, Sequences of proteins of immunological interest, 5 th edition, u.s.department of Health and Human Services, NIH,1991, and later). There are three heavy chain CDRs and three light chain CDRs. Herein, the terms "CDR" and "CDRs" are used to indicate one or more or even all regions, as the case may be, which contain a majority of the amino acid residues responsible for the binding affinity of an antibody to the antigen or epitope it recognizes. To simplify the reading of the present application, the CDRs according to Kabat are not defined. However, it will be apparent to those skilled in the art that the definition of CDRs according to IMGT is used to define CDRs according to Kabat.

In the sense of the present invention, "identity" or "percent identity" between two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment (optimal alignment), which percentage is purely statistical, and the differences between the two sequences are randomly distributed along their length. Comparison of two nucleic acid or amino acid sequences is traditionally performed by comparing the sequences after they have been optimally aligned, which can be performed by fragmentation or by using an "alignment window". In addition to manual comparisons, optimal alignments of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman (1981) [ Ad.App.Math.2:482], the local homology algorithm of Needleman and Wunsch (1970) [ J.mol.biol.48:443], the similar search method of Pearson and Lipman (1988) [ Proc.Natl.Acad.Sci.USA 85:2444], or using Computer Software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group,575Science Dr., Madison, Wis, or by comparison Software BLAST NR or BLAST P).

Percent identity between two sequences is calculated by determining the number of positions at which the amino acid nucleotide or residue is identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window, and multiplying the result by 100 to obtain the percent identity between the two sequences.

For example, the BLAST program "BLAST 2 sequence" (Tatusova et al, "BLAST 2sequences-a new tools for composing protein and nucleotide sequences", FEMS microbiol, 1999, Lett.174: 247-250) may be used by default parameters (especially for the parameters "open gap penalty": 5, and "extended gap penalty": 2; the selected matrix is, for example, the "BLOSUM 62" matrix proposed by the program), which is available on the website http:// www.ncbi.nlm.nih.gov/gorf/bl2. html; the percent identity between the two sequences to be compared is calculated directly by the program.

For amino acid sequences showing at least 80%, preferably 85%, 90%, 95% and 98% identity to a reference amino acid sequence, preferred examples include those comprising the reference sequence, certain modifications being especially deletions, additions or substitutions of at least one amino acid, truncations or elongations. In the case of one or more consecutive or non-consecutive amino acid substitutions, substitutions are preferred in which the substituted amino acid is replaced by an "equivalent" amino acid. Herein, the expression "equivalent amino acid" refers to any amino acid that may be substituted for a structural amino acid without altering the biological activity of the corresponding antibody and those specific examples defined below.

Equivalent amino acids can be determined based on their structural homology to the amino acids substituted by them or the results of comparative tests of biological activity between the various antibodies that may be produced.

As a non-limiting example, table 1 below summarizes possible substitutions that may be made that do not result in a significant change in the biological activity of the corresponding modified antibody; reverse substitution is of course possible under the same conditions.

TABLE 1

A particular aspect of the invention is that the antibody of the ADC does not bind to Insulin Receptor (IR). This aspect is interesting because the antibodies described herein will not have any negative impact on IR, implying insulin metabolism.

In another embodiment, another advantageous aspect of the antibody of the ADC of the invention is that it is capable of binding not only to human IGF-1R, but also to monkey IGF-1R, more particularly to cynomolgus monkey (cynomolgus) IGF-1R. This aspect is also of interest as it will facilitate the toxicity assessment required for clinical trials.

In yet another embodiment, the antibody of the ADC of the invention consists of a monoclonal antibody.

The term "monoclonal antibody" or "Mab" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies of the population are identical except for possible naturally occurring mutations (which may be present in minor amounts). Monoclonal antibodies are highly specific, being directed against a single epitope. Such monoclonal antibodies can be produced by a single clone of a B cell or hybridoma. Monoclonal antibodies may also be recombinant, i.e., produced by protein engineering or chemical synthesis. Monoclonal antibodies can also be isolated from phage antibody libraries. Furthermore, in contrast to preparations of polyclonal antibodies, which typically include a variety of antibodies directed against a variety of determinants or epitopes, each monoclonal antibody is directed against a single epitope of the antigen.

Monoclonal antibodies herein include murine, chimeric and humanized antibodies, as described below.

The antibody is preferably derived from a hybridoma of murine origin deposited at the French national collection of microorganisms (CNCM, pasteur institute, red doctor street No.25, paris 15 region 75724, france) obtained by fusing splenocytes/lymphocytes of Balb/C immunized mice with cells of the myeloma Sp 2/O-Ag 14 cell line.

In one embodiment, the IGF-1R antibody of the ADC of the invention consists of a murine antibody, then called m [ the name of the antibody ].

In one embodiment, the IGF-1R antibody consists of a chimeric antibody, and is then referred to as c [ the name of the antibody ].

In one embodiment, the IGF-1R antibody consists of a humanized antibody, which is then referred to as hz [ the name of the antibody ].

For the avoidance of doubt, in the following specification, the expressions "IGF-1R antibody" and "[ name of antibody ]" are similar and include (without contrary indication) murine, chimeric and humanized forms of said IGF-1R antibody or said "[ name of antibody ]". When necessary, the prefix m- (murine), c- (chimeric) or hz- (humanized) is used.

For greater clarity, table 2 below sets forth CDR sequences defined according to IMGT for preferred antibodies.

TABLE 2

It will be apparent to those skilled in the art that any combination of 6 CDRs as described above should be considered part of the present invention.

It can be observed from this table 2 that all antibodies described herein have the same sequence for CDR-H2, CDR-H3 and CDR-L2, a property that is of particular interest as described above.

A particular aspect relates to an ADC wherein the antibody is a murine antibody, characterised in that said antibody further comprises light and heavy chain constant regions derived from an antibody of a species heterologous to mouse, especially to human.

Another particular aspect relates to an ADC wherein the antibody is a chimeric (c) antibody, characterized in that said antibody further comprises light and heavy chain constant regions derived from an antibody of a species heterologous to mouse, in particular to human.

Chimeric antibodies are antibodies that contain the natural variable regions (light and heavy chains) of an antibody derived from a given species, as well as the constant regions of the light and heavy chains of an antibody of a species heterologous to the given species.

Chimeric antibodies can be prepared by techniques using recombinant genetics. For example, chimeric antibodies can be produced by cloning recombinant DNA containing a promoter and sequences encoding the variable region of a non-human monoclonal antibody, particularly murine, and sequences encoding the constant region of an antibody of a heterologous species, preferably human. The chimeric antibody of the ADC according to the present invention encoded by one such recombinant gene may be, for example, a mouse-human chimera, the specificity of which is determined by the variable region derived from murine DNA and the isotype of which is determined by the constant region derived from human DNA.

In a preferred but non-limiting embodiment, the antibody of the ADC of the invention is selected from the group consisting of:

a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.13 or any sequence showing at least 80% identity with SEQ ID No.13 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11;

b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.14 or any sequence showing at least 80% identity to SEQ ID No.14 and three light chain CDRs of sequences SEQ ID nos. 10, 5 and 11;

c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.15 or any sequence showing at least 80% identity to SEQ ID No.15 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 12;

d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.16 or any sequence showing at least 80% identity to SEQ ID No.16 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11; and

e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.17 or any sequence showing at least 80% identity to SEQ ID No.17 and three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12.

By "any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID nos. 13 to 17" is meant a sequence showing the three heavy chain CDRs SEQ ID nos.1, 2 and 3, and furthermore showing at least 80%, preferably 85%, 90%, 95% and 98% identity with the full sequence SEQ ID nos. 13 to 17 outside the sequences corresponding to the CDRs (i.e. SEQ ID nos.1, 2 and 3).

In another preferred but non-limiting embodiment, the antibody of the ADC of the invention is selected from the group consisting of:

a) an antibody comprising a light chain variable domain of sequence SEQ ID No.18 or any sequence showing at least 80% identity to SEQ ID No.18 and three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3;

b) an antibody comprising a light chain variable domain of sequence SEQ ID No.19 or any sequence showing at least 80% identity with SEQ ID No.19 and three heavy chain CDRs of sequences SEQ ID Nos. 7,2 and 3;

c) an antibody comprising a light chain variable domain of sequence SEQ ID No.20 or any sequence showing at least 80% identity to SEQ ID No.20 and three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3;

d) an antibody comprising a light chain variable domain of sequence SEQ ID No.21 or any sequence showing at least 80% identity to SEQ ID No.21 and three heavy chain CDRs of sequences SEQ ID nos. 8, 2 and 3; and

e) an antibody comprising a light chain variable domain of sequence SEQ ID No.22 or any sequence showing at least 80% identity with SEQ ID No.22 and three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3.

By "any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID nos. 18 to 22" is meant a sequence showing the three light chain CDRs SEQ ID nos. 4, 5 and 6, respectively, and furthermore showing at least 80%, preferably 85%, 90%, 95% and 98% identity with the full sequence SEQ ID nos. 18 to 22 outside the sequences corresponding to the CDRs, i.e. SEQ ID nos. 4, 5 and 6.

One embodiment of the invention relates to an ADC, wherein Ab is an antibody selected from the group consisting of:

a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.13 or any sequence showing at least 80% identity to SEQ ID No.13 and a light chain variable domain of sequence SEQ ID No.18 or any sequence showing at least 80% identity to SEQ ID No. 18;

b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.14 or any sequence showing at least 80% identity to SEQ ID No.14 and a light chain variable domain of sequence SEQ ID No.19 or any sequence showing at least 80% identity to SEQ ID No. 19;

c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.15 or any sequence showing at least 80% identity to SEQ ID No.15 and a light chain variable domain of sequence SEQ ID No.20 or any sequence showing at least 80% identity to SEQ ID No. 20;

d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.16 or any sequence showing at least 80% identity to SEQ ID No.16 and a light chain variable domain of sequence SEQ ID No.21 or any sequence showing at least 80% identity to SEQ ID No. 21; and

e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.17 or any sequence exhibiting at least 80% identity to SEQ ID No.17 and a light chain variable domain of sequence SEQ ID No.22 or any sequence exhibiting at least 80% identity to SEQ ID No. 22.

The chimeric antibodies described herein may also be characterized by constant domains, more particularly, the chimeric antibodies may be selected from or designed such as, but not limited to, IgG1, IgG2, IgG3, IgM, IgA, IgD, or IgE. More preferably, in the context of the present invention, the chimeric antibody is IgG1 or IgG 4.

One embodiment of the invention relates to an ADC wherein Ab is a chimeric antibody comprising variable domains VH and VL as described above in the form of IgG 1. More preferably, the chimeric antibody comprises a constant domain of the VH of sequence SEQ ID No.43 and a kappa domain of the VL of sequence SEQ ID No. 45.

One embodiment of the invention relates to an ADC wherein Ab is a chimeric antibody comprising variable domains VH and VL as described above in the form of IgG 4. More preferably, the chimeric antibody comprises a constant domain of the VH of sequence SEQ ID No.44 and a kappa domain of the VL of sequence SEQ ID No. 45.

a) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.23 or any sequence showing at least 80% identity to SEQ ID No.23 and a light chain of sequence SEQ ID No.28 or any sequence showing at least 80% identity to SEQ ID No. 28;

b) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.24 or any sequence showing at least 80% identity to SEQ ID No.24 and a light chain of sequence SEQ ID No.29 or any sequence showing at least 80% identity to SEQ ID No. 29;

c) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.25 or any sequence showing at least 80% identity to SEQ ID No.25 and a light chain of sequence SEQ ID No.30 or any sequence showing at least 80% identity to SEQ ID No. 30;

d) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.26 or any sequence showing at least 80% identity to SEQ ID No.26 and a light chain of sequence SEQ ID No.31 or any sequence showing at least 80% identity to SEQ ID No. 31; and

e) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.27 or any sequence showing at least 80% identity to SEQ ID No.27 and a light chain of sequence SEQ ID No.32 or any sequence showing at least 80% identity to SEQ ID No. 32.

For greater clarity, table 3 below illustrates the sequences of VH and VL, respectively, for preferred chimeric antibodies.

TABLE 3

Another particular aspect of the invention relates to an ADC wherein "Ab" is a humanized antibody characterized by constant regions derived from the light and heavy chains of a human antibody which are lambda or kappa and gamma-1, gamma-2 or gamma-4 regions, respectively.

"humanized antibody" refers to an antibody that contains CDR regions derived from an antibody of nonhuman origin, the remainder of the antibody molecule being derived from one (or several) human antibodies. In addition, some backbone fragment residues (referred to as FR) can be modified to retain binding affinity.

Humanized antibodies or fragments thereof can be prepared by techniques known to those skilled in the art. Such humanized antibodies are preferably used in methods involving in vitro diagnosis or in vivo prophylactic and/or therapeutic treatment. Other humanization techniques are also known to the person skilled in the art, such as the "CDR grafting" technique described by PDL in patents EP 0451216, EP 0682040, EP 0939127, EP 0566647 or US 5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761. U.S. Pat. Nos. 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 may also be cited.

As a particular embodiment of the invention, and as will be illustrated in more detail in the examples below, an antibody consisting of hz208F2 is described herein. Such humanization may also be applied to other antibody portions of the invention.

In a preferred embodiment, the antibody of the ADC according to the invention comprises a heavy chain variable domain (VH) having:

i) CDR-H1, CDR-H2 and CDR-H3 of sequences SEQ ID Nos. 7,2 and 3, respectively, and

ii) FR1, FR2 and FR3 derived from human germline IGHV1-46 x 01(SEQ ID No.46), and

iii) FR4 derived from human germline IGHJ4 x 01(SEQ ID No. 48).

In a preferred embodiment, the antibody of the ADC according to the invention comprises a light chain variable domain (VL) having:

i) CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ ID Nos. 9, 5 and 11, respectively, and

ii) FR1, FR2 and FR3 derived from the human germline IGKV1-39 x 01(SEQ ID No.47), and

iii) FR4 derived from human germline IGKJ4 x 01(SEQ ID No. 49).

In a preferred but non-limiting embodiment of the invention, the antibody comprises:

In one embodiment, the antibody of the ADC according to the invention comprises a heavy chain variable domain (VH) of sequence SEQ ID No.33 and a light chain variable domain (VL) of sequence SEQ ID No. 35. The humanized antibody will be referred to hereinafter as hz208F2 ("variant 1" or "var.1").

In another embodiment, the antibody of the ADC according to the invention comprises the heavy chain variable domain (VH) of sequence SEQ ID No.33, wherein said sequence SEQ ID No.33 comprises at least 1 back mutation selected from the group consisting of residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95.

The expression "back-mutation" or "back-mutation" refers to a mutation or substitution in which a human residue present in the germline is replaced with the corresponding residue originally present in the murine sequence.

In another embodiment, the antibody of the ADC according to the invention comprises the heavy chain variable domain (VH) of sequence SEQ ID No.33, wherein said sequence SEQ ID No.33 comprises 2,3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 back-mutations selected from residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95.

For greater clarity, table 4 below sets forth preferred back mutations.

TABLE 4

Residue number

20

34

35

38

48

50

59

61

Mouse

M

I

Y

K

L

W

K

N

Human being

V

M

H

R

M

I

S

A

Residue number

62

70

72

74

76

77

79

82

95

Mouse

E

L

A

K

S

N

A

F

Human being

Q

M

R

T

S

V

E

Y

In one embodiment, the antibody of the ADC according to the invention comprises the light chain variable domain (VL) of sequence SEQ ID No.35, wherein said sequence SEQ ID No.35 comprises at least 1 back mutation selected from the group consisting of residues 22, 53, 55, 65, 71, 72, 77 and 87.

In one embodiment, the antibody of an ADC according to the invention comprises the light chain variable domain (VL) of sequence SEQ ID No.35, wherein said sequence SEQ ID No.35 comprises 2,3, 4, 5,6, 7 or 8 back-mutations selected from residues 22, 53, 55, 65, 71, 72, 77 or 87.

In another embodiment, an antibody to an ADC according to the invention comprises:

a) a heavy chain variable domain (VH) of sequence SEQ ID No.33, wherein said sequence SEQ ID No.33 comprises at least 1 back mutation selected from the group consisting of residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95; and

b) a light chain variable domain (VL) of sequence SEQ ID No.35 wherein said sequence SEQ ID No.35 comprises at least 1 back mutation selected from the group consisting of residues 22, 53, 55, 65, 71, 72, 77 and 87.

For greater clarity, table 5 below sets forth preferred back mutations.

TABLE 5

Residue number

22

53

55

65

71

72

77

87

Mouse

S

R

H

R

Y

S

N

F

Human being

T

S

Q

S

F

T

S

Y

In one such embodiment, the antibody of the ADC according to the invention comprises all of the back-mutations mentioned above and corresponds to an antibody comprising a heavy chain variable domain (VH) of sequence SEQ ID No.34 and a light chain variable domain (VL) of sequence SEQ ID No. 36. The humanized antibody will be referred to hereinafter as hz208F2 ("variant 3" or "var.3").

In another embodiment, the invention also encompasses all humanized forms comprised between variant 1 and variant 3. In other words, the antibody according to the invention corresponds to an antibody comprising a heavy chain variable domain (VH) of the "consensus" sequence SEQ ID No.41 and a light chain variable domain (VL) of the "consensus" sequence SEQ ID No. 42. In general, the humanized antibody will be referred to hereinafter as hz208F2 ("variant 2" or "var.2").

a) an antibody comprising the heavy chain variable domain of sequence SEQ ID No.33 or any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.33 and the three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11; and

b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.34 or any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.34 and three light chain CDRs of sequences SEQ ID nos. 9, 5 and 11.

By "any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.33 or 34" is meant a sequence showing the three heavy chain CDRs SEQ ID No.1, 2 and 3, and furthermore showing at least 80%, preferably 85%, 90%, 95% and 98% identity with the full sequence SEQ ID No.33 or 34 outside the sequences corresponding to the CDRs (i.e. SEQ ID nos.1, 2 and 3).

If it is not indicated in the relevant paragraphs of the specification that at least 80% of the sequence is shown by any sequence or by being associated with a particular sequence, it must be understood that the sequence shows at least 80%, preferably 85%, 90%, 95% and 98% identity to the reference sequence. Whether or not these sequences contain CDR sequences, it means sequences that show at least these CDRs identical to the CDRs of the reference sequence, with 80%, preferably 85%, 90%, 95% and 98% identity to the entire sequence, requiring the calculation of the remaining sequences that lie outside the sequences corresponding to these CDRs.

In a preferred but non-limiting embodiment, the antibody of the invention is selected from the group consisting of:

a) an antibody comprising the light chain variable domain of sequence SEQ ID No.35 or any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.35 and the three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3; and

b) an antibody comprising the light chain variable domain of sequence SEQ ID No.36 or any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.36 and the three heavy chain CDRs of sequences SEQ ID Nos. 7,2 and 3.

By "any sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.35 or 36" is meant a sequence showing the three light chain CDRs SEQ ID No.4, 5 and 6, and furthermore showing at least 80%, preferably 85%, 90%, 95% and 98% identity with the full sequence SEQ ID No.35 or 36 outside the sequences corresponding to the CDRs, i.e. SEQ ID nos. 4, 5 and 6.

The humanized antibodies described herein may also be characterized by constant domains, more particularly, the humanized antibodies may be selected from or designed such as, but not limited to, IgG1, IgG2, IgG3, IgM, IgA, IgD, or IgE. More preferably, in the context of the present invention, the humanized antibody is IgG1 or IgG 4.

One embodiment of the invention relates to an ADC wherein the "Ab" is a humanized antibody comprising variable domains VH and VL as described above in the form of IgG 1. More preferably, the humanized antibody comprises a constant domain of the VH of sequence SEQ ID No.43 and a kappa domain of the VL of sequence SEQ ID No. 45.

One embodiment of the invention relates to an ADC wherein the "Ab" is a humanized antibody comprising variable domains VH and VL as described above in the form of IgG 4. More preferably, the humanized antibody comprises a constant domain of the VH of sequence SEQ ID No.44 and a kappa domain of the VL of sequence SEQ ID No. 45.

Yet another embodiment of the invention relates to an ADC, wherein "Ab" is an antibody selected from the group consisting of:

For greater clarity, table 6a below sets forth non-limiting examples of sequences of the VH and VL of variant 1(var.1) and variant 3(var.3) of humanized antibody hz208F 2. It also comprises the consensus sequence of variant 2 (var.2).

TABLE 6a

a) an antibody comprising a heavy chain variable domain of a sequence selected from SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80, and the three light chain CDRs of sequences SEQ ID nos. 9, 5 and 1;

b) an antibody comprising a light chain variable domain of a sequence selected from SEQ ID No.57 or 60 or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.57 or 60, and three heavy chain CDRs of sequences SEQ ID nos. 7,2 and 3; and

c) an antibody comprising a heavy chain variable domain selected from the sequences of SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80, and a light chain variable domain selected from the sequence of SEQ ID nos. 57 or 60 or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity to SEQ ID nos. 57 or 60.

a) an antibody comprising the heavy chain of sequences SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence exhibiting at least 80% identity to SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80, and the light chain of sequence SEQ ID No.57 or any sequence exhibiting at least 80% identity to SEQ ID No. 57; and

b) an antibody comprising the heavy chain of sequences SEQ ID Nos. 56, 64, 68 and 78 or any sequence showing at least 80% identity to SEQ ID Nos. 56, 64, 68 or 78 and the light chain of sequence SEQ ID No.60 or any sequence showing at least 80% identity to SEQ ID No. 60.

Yet another embodiment of the invention relates to an ADC, wherein Ab is an antibody selected from the group consisting of:

a) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.58 or any sequence showing at least 80% identity to SEQ ID No.58 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

b) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.58 or any sequence showing at least 80% identity to SEQ ID No.58 and a light chain of sequence SEQ ID No.61 or any sequence showing at least 80% identity to SEQ ID No. 61;

c) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.63 or any sequence showing at least 80% identity to SEQ ID No.63 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

d) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.65 or any sequence showing at least 80% identity to SEQ ID No.65 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

e) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.65 or any sequence showing at least 80% identity to SEQ ID No.65 and a light chain of sequence SEQ ID No.61 or any sequence showing at least 80% identity to SEQ ID No. 61;

f) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.67 or any sequence showing at least 80% identity to SEQ ID No.67 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

g) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.69 or any sequence showing at least 80% identity with SEQ ID No.69 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity with SEQ ID No. 59;

h) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.69 or any sequence showing at least 80% identity with SEQ ID No.69 and a light chain of sequence SEQ ID No.61 or any sequence showing at least 80% identity with SEQ ID No. 61;

i) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.71 or any sequence showing at least 80% identity to SEQ ID No.71 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

j) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.73 or any sequence showing at least 80% identity to SEQ ID No.73 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

k) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.75 or any sequence showing at least 80% identity to SEQ ID No.75 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

l) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.77 or any sequence showing at least 80% identity to SEQ ID No.77 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

m) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.79 or any sequence showing at least 80% identity to SEQ ID No.79 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59;

n) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.79 or any sequence showing at least 80% identity with SEQ ID No.79 and a light chain of sequence SEQ ID No.61 or any sequence showing at least 80% identity with SEQ ID No. 61; and

o) an antibody comprising or consisting of a heavy chain of sequence SEQ ID No.81 or any sequence showing at least 80% identity to SEQ ID No.81 and a light chain of sequence SEQ ID No.59 or any sequence showing at least 80% identity to SEQ ID No. 59.

In other words, the invention relates to an ADC wherein Ab is an antibody comprising:

a) a heavy chain of a sequence selected from SEQ ID nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 and 81 or any sequence having at least 80% identity to SEQ ID nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 and 81; and

b) a light chain selected from the sequences of SEQ ID Nos. 59 and 61 or any sequence having at least 80% identity to SEQ ID Nos. 59 and 61.

For greater clarity, table 6b below sets forth a non-limiting example of the sequences of VH and VL (variable domains and full length) of different variants of humanized antibody hz208F 2.

TABLE 6b

Another aspect of the invention is an ADC, wherein Ab is an antibody selected from the group consisting of: i) antibodies produced by hybridomas I-4757, I-4773, I-4775, I-4736, or I-4774 deposited in CNCM, Pasteur, France, at 30/5/2013, 26/6/2013, 24/2013, and 26/6/2013, respectively; or ii) an antibody that competes with the antibody of i) for binding to IGF-1R; or iii) an antibody that binds to the same epitope of IGF-1R as the antibody of i).

Indeed, described herein are murine hybridomas selected from the hybridomas I-4757, I-4773, I-4775, I-4736 and I-4774, deposited at CNCM, the Pasteur institute of France, on 5/30, 6/26, 2013, 4/24 and 6/26, 2013, respectively.

Also described are ex vivo nucleic acids encoding antibodies or antigen-binding fragments thereof according to the invention.

The terms "nucleic acid", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in this specification, refer to a precise nucleotide sequence, modified or unmodified, which defines a fragment or region of a nucleic acid, which contains or does not contain non-natural nucleotides, and which is double-stranded DNA, single-stranded DNA or a transcript of said DNA.

These sequences have been isolated and/or purified, i.e. they have been sampled, directly or indirectly, e.g. by copying, the environment of which has been at least partially modified. Also mentioned herein are ex vivo nucleic acids obtained by recombinant genetics, e.g., using host cells, or by chemical synthesis.

Vectors comprising nucleic acids encoding the antibodies or antigen-binding fragments thereof of the ADCs according to the invention are also described, as are cloning and/or expression vectors containing such nucleotide sequences.

The vector preferably contains elements which allow for expression and/or secretion of the nucleotide sequence in a given host cell. Thus, the vector may contain a promoter, translation initiation and termination signals, and appropriate transcriptional regulatory regions. It must be able to be maintained in a stable manner in the host cell and may optionally have specific signals which specify the secretion of the translated protein. These various elements are selected and optimized by the skilled person depending on the host cell used. For this purpose, the nucleotide sequence may be inserted into a self-replicating vector within the chosen host, or be an integrating vector for the chosen host.

The vector is, for example, a plasmid or a virus-derived vector. They are used to transform host cells to clone or express the nucleotide sequences of the present invention.

Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods such as lipofection, electroporation, conjugation, heat shock or chemical methods.

These ex vivo host cells are transformed by or comprise a vector as described above.

The host cell may be selected from prokaryotic or eukaryotic systems, such as bacterial cells, but may also be selected from yeast cells or animal cells, in particular mammalian cells (except human). Insect or plant cells may also be used.

Also disclosed is a method for producing an antibody or antigen-binding fragment thereof of an ADC according to the invention, wherein the method comprises the steps of:

a) culturing a host cell as disclosed above in a medium under suitable culture conditions; and

b) recovering the antibody thus produced from the culture medium or from the cultured cells.

The transformed cells are used in a method for producing a recombinant antibody for an ADC according to the invention. Also included in the present specification are methods for producing recombinant forms of the antibodies using vectors and/or cells transformed with vectors as disclosed above. Preferably, the cells transformed with the vector as described above are cultured under conditions which allow the expression of the above-mentioned antibody and the recovery of said antibody.

As already mentioned, the host cell may be selected from prokaryotic or eukaryotic systems. In particular, in such prokaryotic or eukaryotic systems, nucleotide sequences that promote secretion can be identified. Thus, vectors carrying such sequences as disclosed above may advantageously be used for the production of recombinant proteins to be secreted. In fact, the presence of these recombinant proteins of interest in the supernatant of the cell culture, rather than within the host cell, will facilitate the purification of said recombinant proteins.

Antibodies to the ADCs of the present invention may also be prepared by chemical synthesis. One such preparation method is also an object of the present invention. The person skilled in the art is aware of methods for chemical synthesis, such as solid phase techniques or partial solid phase techniques by condensation of fragments or by conventional synthesis in solution. Mention may also be made of polypeptides obtained by chemical synthesis and capable of containing the corresponding unnatural amino acid.

The invention also includes antibodies that are obtainable by the methods described above.

According to a particular aspect, the invention relates to an ADC wherein AB is an antibody or antigen-binding fragment thereof as described above, for use as an addressing vector (addressing vehicle) for delivering a cytotoxic agent at a host target site consisting of an epitope located at the N-terminus of an IGF-1R, preferably an IGF-1R extracellular domain, more preferably a human IGF-1R (SEQ ID No.50), still more preferably a human IGF-1R extracellular domain (SEQ ID No.51), still more preferably to a human IGF-1R extracellular domain (SEQ ID No.52) or any native variant sequence thereof.

In a preferred embodiment, the host target site is a target site of a mammalian cell, more preferably a target site of a human cell, more preferably a target site of a cell that naturally expresses or expresses IGF-1R by genetic recombination.

In further embodiments, the host target site is a target site of a cell of a patient, preferably a human, suffering from a cancer, preferably an IGF-1R-expressing cancer or an IGF-1R-associated cancer.

Cancers expressing IGF-1R or IGF-1R-associated cancers include in particular cancers in which tumor cells express or overexpress all or part of the IGF-1R on their surface.

II-medicine (D)

The pharmaceutical moiety according to the invention has the following formula (II)

Wherein:

-R₂is COOH, COOCH₃Or thiazolyl (such as thiazol-2-yl),

-R₃is H or (C)₁-C₆) Alkyl radicals (e.g. methyl), especially (C)₁-C₆) An alkyl group, a carboxyl group,

-R₉is H or (C)₁-C₆) An alkyl group (such as a methyl group),

-m is an integer between 1 and 8, and

the wavy line indicates the point of connection to L.

The term "alkyl" as used herein refers to a straight or branched, saturated hydrocarbon chain. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl may be mentioned.

In the present invention, "(C)_x-C_y) Alkyl "refers to an alkyl chain as defined above containing x to y carbon atoms. Thus, (C)₁–C₆) Alkyl radicalIs an alkyl chain having 1 to 6 carbon atoms.

(C₁–C₆) Alkyl is advantageously (C)₁–C₄) Alkyl, preferably (C)₁–C₂) An alkyl group.

In the compounds of the invention, one particularly preferred class of drug moieties corresponds to those of formula (II) wherein R₂Represents a COOH group.

Another particularly preferred class of moieties corresponds to those of formula (II) wherein R₂Is thiazole (in particular thiazol-2-yl).

Another particularly preferred class of moieties corresponds to those of formula (II) wherein R₂Is COOMe.

According to a particular embodiment of the invention, R₂More particularly a COOH, COOMe or thiazol-2-yl group.

According to a first preferred embodiment, R₂Is COOH.

According to a second preferred embodiment, R₂Is COOMe.

R₃Particularly represents (C)₁-C₆) Alkyl, advantageously methyl.

m is an integer between 1 and 8, in particular between 1 and 6, advantageously between 1 and 4, preferably 1 or 2.

In a preferred embodiment, R₂Is COOH, R₃Is methyl and m is 1 or 2.

Of the drug moieties of the present invention, one particularly preferred class of drug moieties corresponds to those of formula (II) wherein R₉Is methyl or hydrogen.

In a preferred embodiment:

-R₂is COOH, R₃Is methyl, R₉Is methyl, and m is 1 or 2, or

-R₂Is COOH, R₃Is methyl, R₉Is hydrogen, and m is 1 or 2.

According to a preferred embodiment, NR₉The radical being located on the benzene ring opposite (CH)₂)_mPara to the group.

Advantageously, the drug moiety is selected from the following moieties:

preparation of the drug (of formula DH):

the medicaments may be prepared using the general methods described in the synthetic schemes below, optionally supplemented with any standard procedures described in the literature or well known to those skilled in the art, or described in the examples of the experimental section herein, as required.

Scheme 1 illustrates the first general method that can be used to prepare a drug. In the above formula, R₁＝H，R₂And R₃As defined in formula II above, R₄RepresentsR_4aRepresents R as defined above, optionally in protected form₄A group, and G is a protecting group。

The first step consists of the condensation of compound (II) protected on its amine function by a protecting group G, with a compound (III). X may represent a leaving group such as chloro. In this case, the first step consists of the reaction of an acid chloride (acid chloride) with an amine. The reaction can be carried out using methods and techniques well known to those skilled in the art. In a particularly preferred method, in an organic or inorganic base, e.g. Et₃N、iPr₂NEt, pyridine, NaH, Cs₂CO₃、K₂CO₃The two entities are reacted in a solvent such as THF, dichloromethane, DMF, DMSO, especially at a temperature of-20 ℃ to 100 ℃. X may also be a hydroxyl group (OH). In this case, the first step is a condensation reaction of carboxylic acid (II) and amine (III). The reaction may be carried out according to methods and techniques well known to those skilled in the art. In a particularly preferred method, the two entities are reacted in the presence of a coupling agent such as 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide (EDC), 3-hydroxy-1, 2, 3-benzotriazin-4 (3H) -one, a tertiary amine such as diisopropylethylamine in a polar aprotic solvent such as dichloromethane or DMF, especially at a temperature of-15 ℃ to 40 ℃. In another particularly preferred process, the two entities are reacted in the presence of diethyl cyanophosphate (DEPC), a tertiary amine such as triethylamine, in a polar aprotic solvent such as dichloromethane or DMF at a temperature of from-15 ℃ to 40 ℃. Another particularly recommended method consists of: the two entities are reacted in the presence of O- (7-azabenzotriazol-1-yl) -1,1,3, 3-tetramethyl-urea Hexafluorophosphate (HATU), a tertiary amine such as diisopropylethylamine in a polar aprotic solvent such as dichloromethane or DMF at a temperature of-15 ℃ to 100 ℃.

Using techniques well known to those skilled in the art (Protective Groups in organic Synthesis, T.W.Greene, John Wiley&After deprotection of the intermediate by Sons,2006 and Protecting Groups, p.j.kocienski, Thieme Verlag,1994, compound (IV) may be condensed with compound (V) according to the methods and techniques described above to yield the compound after the deprotection step (c: (c) (r))VI). This compound may then, after condensation with intermediate (VII) and optional deprotection, lead to the formation of a drug. Compound (VI) may also be coupled with compound (VII '), wherein R'₃Is R₃In particular R protected by a protecting group₃A group. Group R'₃Deprotection to R₃The coupling can then be carried out according to the same procedure as described previously.

Scheme 2 illustrates a second general method that can be used to prepare a drug. In the above formula, G is a protecting group, R₁＝H，R₂、R₃And R_4aAs defined above, and R_4bRepresents

In a first step, compound (IX) protected on its amine function by a protecting group G is condensed with compound (VI). X may represent a leaving group, such as chloro. In this case, the first step consists of the reaction of the acid chloride with the amine. The reaction can be carried out using methods and techniques well known to those skilled in the art. In a particularly preferred method, in an organic or inorganic base, e.g. Et₃N、iPr₂NEt, pyridine, NaH, Cs₂CO₃、K₂CO₃The two entities are reacted in a solvent such as THF, dichloromethane, DMF, DMSO, especially at a temperature of-20 ° to 100 ℃. X may also represent a hydroxyl group. In this case, the first step is the condensation of the carboxylic acid (IX) with the amine (VI). The reaction may be carried out according to methods and techniques well known to those skilled in the art. In a particularly preferred process, the two are reacted in the presence of 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide (EDC), 3-hydroxy-1, 2, 3-benzotriazin-4 (3H) -one, a tertiary amine such as diisopropylethylamine in a polar aprotic solvent such as dichloromethane or DMF, especially at a temperature of-15 ℃ to 40 ℃The seed entity is reacted. In another particularly preferred process, the two entities are reacted in the presence of diethyl cyanophosphate (DEPC), a tertiary amine such as triethylamine, in a polar aprotic solvent such as dichloromethane or DMF, especially at temperatures of-15 ℃ to 40 ℃.

After deprotection of the intermediate, the resulting compound (VIII) is reacted with R using techniques well known to the skilled artisan₄Y can produce a drug after reaction. In this case, Y is a leaving group such as Cl, Br, I, OSO₂CH₃、OSO₂CF₃Or O-tosyl. In organic or inorganic bases, e.g. Et₃N、iPr₂NEt、NaH、Cs₂CO₃、K₂CO₃The reaction is carried out in a polar anhydrous solvent such as dichloromethane, THF, DMF, DMSO, especially at a temperature of-20 ° to 100 ℃. In another particularly preferred process, the compound (VIII) is reacted with a compound of the formula R_4b-CHO of an aldehyde, wherein R_4bCorresponds to R₄A precursor of (2). In this case, the reaction is carried out in a reducing agent such as NaBH₄、NaBH₃CN、NaBH(OAc)₃In a polar solvent such as 1, 2-dichloroethane, dichloromethane, THF, DMF, MeOH, optionally in the presence of titanium (IV) isopropoxide, at a pH which can be controlled by addition of an acid such as acetic acid, in particular at a temperature of from-20 ℃ to 100 ℃.

In the foregoing synthetic schemes, one drug may yield another drug after an additional reaction step such as saponification (e.g., using methods well known to the skilled artisan), whereby the R representing the ester (COOMe)₂Conversion of the group into R representing a carboxylic acid (COOH)₂A group.

If it is desired to isolate a drug containing at least one base functionality in the state of an acid addition salt, it is possible to treat the free base (containing at least one base functionality) of the drug with a suitable acid, preferably in an equivalent amount. A suitable acid may in particular be trifluoroacetic acid.

III-Joint (L)

In the present invention, "linker", "linker unit", "L" or "link" refers to a chemical moiety comprising a covalent bond or chain of atoms, which covalently links at least one drug to an antibody.

Linkers can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azides (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-reactive fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating cytotoxic agents to addressing systems. Other cross-linking agents may be BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC, and thio-SMPB and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

The linker may be "non-cleavable" or "cleavable".

In a preferred embodiment, it consists in that the "cleavable linker" facilitates the release of the drug in the cell. For example, an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide containing linker may be used. In a preferred embodiment, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug from the antibody in an intracellular environment.

For example, in some embodiments, the linker can be cleaved by a cleaving agent that is present in the intracellular environment (e.g., within lysosomes or endosomes or the cytosol). The linker may be, for example, a peptidyl linker, which is cleaved by an intracellular peptidase or protease (including, but not limited to, lysosomal proteases or endosomal proteases). Typically, a peptidyl linker comprises at least two consecutive amino acids or at least three consecutive amino acids, or is at least two amino acids long or at least three amino acids long. Lytic agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in release of the active drug within the target cell. For example, a peptidyl linker (e.g., a linker comprising or being Phe-Leu or Gly-Phe-Leu-Gly) that is cleavable by the thiol-dependent protease cathepsin B, which is highly expressed in cancerous tissues, can be used. In particular embodiments, the peptidyl linker cleavable by an intracellular protease comprises either Val-Cit or Phe-Lys. One advantage of using intracellular proteolytic drug release is that the drug is often attenuated when conjugated and the serum stability of the conjugate is generally high.

In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH sensitive linker is hydrolysable under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) may be used. The linker is relatively stable under neutral pH conditions (such as those in blood), but is unstable below pH 5.5 or 5.0 (close to the pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to the drug via an acylhydrazone bond).

in other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker) various disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate), SPDB (N-succinimidyl-3- (2-pyridyldithio) butyrate), and SMTP (N-succinimidyl-oxycarbonyl- α -methyl- α - (2-pyridyl-dithio) toluene).

In certain preferred embodiments, the linker unit may have the general formula:

-(T)_a-(W)_w-(Y)_y-

wherein:

t is a stretcher unit;

a is 0 or 1;

w is an amino acid unit;

w is an integer between 0 and 12;

y is a spacer unit (spacer unit);

y is 0,1 or 2.

The stretcher unit (T), when present, links the antibody to the amino acid unit (W), when present, or to the spacer unit, when present, or directly to the drug. Useful functional groups include sulfhydryl, amino, hydroxyl, anomeric (anomeric) hydroxyl and carboxyl groups of carbohydrates, which may be present naturally on the antibody or via chemical modification. Suitable functional groups are mercapto and amino. Sulfhydryl groups (if present) may be generated by reduction of intramolecular disulfide bonds of the antibody. Alternatively, sulfhydryl groups may be generated by reaction of the amino group of a lysine moiety of an antibody with 2-iminothiolane or other sulfhydryl generating reagent. In particular embodiments, the antibody is engineered to carry one or more lysines. More preferably, the antibody may be engineered to carry one or more cysteines (see ThioMab).

In certain specific embodiments, the stretcher subunit forms a bond with a sulfur atom of the antibody. The sulfur atom may be derived from the thiol (-SH) group of the reduced antibody.

In certain other embodiments, the stretcher unit is attached to the antibody via a disulfide bond between a sulfur atom of the antibody and a sulfur atom of the stretcher unit.

In other embodiments, the reactive group of the stretcher contains a reactive site that can react with an amino group of an antibody. The amino group may be an amino group of arginine or lysine. Suitable amine reactive sites include, but are not limited to, activated esters (e.g., succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

In another aspect, the reactive functional group of the stretcher comprises a reactive site that reacts with a modified carbohydrate group that may be present on the antibody. In a specific embodiment, the antibody is enzymatically glycosylated to provide a carbohydrate moiety, or the antibody is naturally glycosylated. Carbohydrates can be oxidized mildly with reagents such as sodium periodate and the resulting carbonyl units of the oxidized carbohydrates can be condensed with stretchers that contain functional groups such as hydrazides, oximes, reactive amines, hydrazines, thiosemicarbazides, hydrazines carboxylates, or aryl hydrazides.

According to a particular embodiment, the stretching subunit has the formula:

wherein

L₂Is (C)₄-C₁₀) Cycloalkyl-carbonyl group, (C)₂-C₆) Alkyl or (C)₂-C₆) Alkyl-carbonyl (cycloalkyl or alkyl moiety is attached to the nitrogen atom of the maleimide moiety),

the asterisks indicate the point of attachment to the amino acid unit (if present), to the spacer unit (if present), or to the drug D, and

the wavy line indicates the point of attachment to antibody Ab.

In the present invention, "(C)₄-C₁₀) Cycloalkyl "refers to a hydrocarbon ring having 4 to 10 carbon atoms, including but not limited to cyclopentyl, cyclohexyl, and the like.

L₂May advantageously be (C)₂-C₆) Alkyl-carbonyl, such as pentyl-carbonyl of the formula:

wherein

Asterisks indicate the point of attachment to the amino acid unit (if present), to the spacer unit (if present), or to drug D; and

the wavy line indicates the point of attachment to the nitrogen atom of the maleimide moiety.

The amino acid unit (W) (when present) will stretch the subunit (T) (if present) or will link the antibody to the spacer unit (Y) (if a spacer unit is present), or to the drug (if a spacer unit is not present).

As mentioned above, (W)_wAbsent (w ═ 0) or may be dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide units, where the amino acids forming the peptides may be different from each other.

Thus, it can be represented by the following formula (W)_w：(W1)_w1(W2)_w2(W3)_w3(W4)_w4(W5)_w5Wherein each W1 to W5 independently of each other represents an amino acid unit and each W1 to W5 is 0 or 1.

In some embodiments, an amino acid unit (W)_wMay comprise amino acid residues (such as those naturally occurring), andminor (minor) amino acids and non-naturally occurring amino acid analogs, such as citrulline.

Amino acid unit (W)_wThe amino acid residues of (a) include, but are not limited to, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine protected or unprotected with acetyl or formyl, arginine protected or unprotected with tosyl or nitro, histidine, ornithine protected with acetyl or formyl, and citrulline. Exemplary amino acid linker components preferably include di-, tri-, tetra-or pentapeptides, especially di-or tripeptides.

Exemplary dipeptides include: Val-Cit, Ala-Val, Ala-Ala, Val-Ala, Lys-Lys, Cit-Cit, Val-Lys, Ala-Phe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N⁹-tosyl-Arg、Phe-N⁹-nitro-Arg.

Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Phe-Phe-Lys, Gly-Gly-Gly, D-Phe-Phe-Lys, Gly-Phe-Lys.

Exemplary tetrapeptides include: Gly-Phe-Leu-Gly (SEQ ID NO.53), Ala-Leu-Ala-Leu (SEQ ID NO. 54).

Exemplary pentapeptides include: Pro-Val-Gly-Val-Val (SEQ ID NO. 55).

According to a particular embodiment, (W)_wIt may be a dipeptide (i.e. w ═ 2) such as Val-Cit, or a linker lacking an amino acid unit (w ═ 0). When a linker lacks an amino acid unit, it preferably also lacks a spacer unit.

According to a preferred embodiment, W ═ 0 (i.e., (W)_wIs a single bond) or W ═ 2 (i.e., (W)_wIs a dipeptide), therefore (W)_wMay be selected from:

and in particular Val-Cit,

wherein

Asterisks indicate the points of attachment to the spacer subunit (if present), or to drug D; and

wavy line representation and L₂The point of connection.

The amino acid linker component may be designed and optimized in terms of its selectivity for cleavage by a particular enzyme, e.g., tumor-associated proteases, cathepsin B, C and D or plasmin protease.

The amino acid units of the linker may be enzymatically cleaved by enzymes (including, but not limited to, tumor-associated proteases) to release the drug.

The amino acid units can be designed and optimized with respect to their enzymatic selectivity for specific tumor-associated proteases. Suitable units are those whose cleavage is catalyzed by proteases, cathepsins B, C and D and plasmin.

The spacer unit (Y) (when present) links the amino acid unit (if present) or the stretcher unit (if present) or the antibody to the drug. There are two general types of spacer elements: self-decomposed (self-immolative) and non-self-decomposed. The non-self-immolative spacer unit is a spacer unit in which some or all of the spacer unit remains bound to the drug after the amino acid unit is cleaved from the enzyme in the antibody-drug conjugate. Examples of spacer units that are not self-immolative include, but are not limited to, (glycine-glycine) spacer units and glycine spacer units. To release the drug, a separate hydrolysis reaction should be performed within the target cell to cleave the glycine-drug unit bond.

In a particular embodiment, the non-self-immolative spacer unit (Y) is Gly.

Alternatively, the antibody-drug conjugate containing a self-immolative spacer unit need not be a separate oneThe hydrolysis step releases the drug. In these embodiments, (Y) is the residue of a p-aminobenzyl alcohol (PAB) unit, which is linked to (W) via the nitrogen atom of the PAB group_wAnd directly linked to the drug via an ester, carbonate, carbamate, or ether group.

Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electrically (electronically) equivalent to the PAB group, such as the residue of a 2-aminoimidazole-5-methanol derivative and the residue of an ortho-or para-aminobenzyl acetal. Spacers which readily undergo cyclization upon hydrolysis of the amide bond may be used, such as substituted and unsubstituted 4-aminobutanoic acid amides, appropriately substituted bicyclo [2.2.1] and bicyclo [2.2.2] ring systems, and 2-aminophenylpropionic acid amide.

In an alternative embodiment, the spacer unit is a branched bis (hydroxymethyl) styrene (BHMS) unit, which may be used to incorporate additional drugs.

In a particular embodiment, the spacer unit (Y) is a PAB-carbonyl group, wherein PAB is(the oxygen of the PAB unit is attached to the carbonyl group) and y ═ 1 or the linker lacks a spacer unit (y ═ 0).

In a particular embodiment, the linker has the following formula (III):

wherein

L₂Is (C)₄-C₁₀) Cycloalkyl-carbonyl group, (C)₂-C₆) Alkyl or (C)₂-C₆) Alkyl-carbonyl (the carbonyl groups of these moieties, when present, are attached to (W)_w)，

W represents an amino acid unit, wherein W represents an integer between 0 and 5,

y is PAB-carbonyl, wherein PAB is(the oxygen of the PAB unit is attached to the carbonyl group) and y is 0 or 1 (preferably y is 0 when w is 0 and is 0 or 1 when w is 1 to 5),

asterisks indicate points of attachment to drug D, and

the wavy line indicates the point of attachment to antibody Ab.

Advantageously, L₂Is (C)₂-C₆) Alkyl-carbonyl, such as pentyl-carbonyl of the formula:

wherein

Star sign of AND (W)_wA point of connection; and

According to a preferred embodiment, the linker L is selected from:

where the asterisks indicate the point of attachment to drug D and the wavy line indicates the point of attachment to antibody Ab.

IV-antibody-drug conjugates (ADC)

In a preferred embodiment, the antibody-drug conjugates of the invention can be prepared by any method known to those skilled in the art, such as, but not limited to, i) reacting a nucleophilic group of an antibody with a bivalent linker reagent and then with a nucleophilic group of a drug, or ii) reacting a nucleophilic group of a drug with a bivalent linker reagent and then with a nucleophilic group of an antibody.

Nucleophilic groups on antibodies include, but are not limited to, N-terminal amine groups, side chain amine groups (e.g., lysine), side chain sulfhydryl groups, and sugar hydroxyl or amino groups when the antibody is glycosylated.

Nucleophilic groups on the drug include, but are not limited to, amine groups, thiol groups, and hydroxyl groups, with amine groups being preferred.

The amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including but not limited to active esters such as NHS esters, HOBt esters, haloformates, and acid halides; alkyl halides and benzyl halides, such as haloacetamides; an aldehyde; a ketone; a carboxyl group; and a maleimide group. The antibody may have a reducible interchain disulfide, i.e., a cysteine bridge. The antibody may be rendered reactive for conjugation to a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Thus, each cysteine bridge will theoretically form two reactive thiol nucleophiles (nucleoophiles). Additional nucleophilic groups can be introduced into the antibody by any reaction known to those skilled in the art. As a non-limiting example, a reactive thiol group may be introduced into an antibody by introducing one or more cysteine residues.

Electrophilic moieties, which can react with nucleophilic substituents on linker reagents, can also be introduced by modifying the antibody to produce antibody-drug conjugates. The sugar of the glycosylated antibody may be oxidized to form an aldehyde or ketone group that may react with the amine group of the linker reagent or drug. The resulting imine Schiff (Schiff) base groups may form stable bonds or may be reduced to form stable amine bonds. In one embodiment, the reaction of the carbohydrate moiety of a glycosylated antibody with galactose oxidase or sodium metaperiodate may produce carbonyl (aldehyde and ketone) groups in the protein that may react with suitable groups on the drug. In another embodiment, a protein containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate resulting in the production of an aldehyde in place of the first amino acid.

In a preferred embodiment, the antibody-drug conjugates of the invention are prepared by preparing a drug-linker moiety and then coupling a nucleophilic group of the antibody (e.g., an SH group of a cysteine moiety) to an electrophilic group of the drug-linker moiety (e.g., a maleimide).

1. Drug-linker

The drug-linker moiety may be prepared by the following coupling:

-coupling the linker to the drug,

-coupling a part of the linker with the drug before completing the synthesis of the linker,

coupling a linker to a part or precursor of the drug before the synthesis of the drug is completed, or

-coupling a part of the linker with a part of the drug or a precursor thereof before completing the synthesis of the linker and the drug.

The coupling reaction between nucleophilic and electrophilic groups is a reaction well known to those skilled in the art.

The nucleophilic group may in particular be an amine group, a thiol group or a hydroxyl group. In a preferred embodiment, it is a primary or secondary amine group.

The electrophilic group can be a carboxylic acid group (COOH) optionally in activated form or an activated carbonate moiety.

By "activated form" of a carboxylic acid is meant a carboxylic acid in which the OH moiety of the COOH functional group has been replaced by an activated Leaving Group (LG) capable of coupling an activated carboxylic acid group with an amino group to form an amide bond and release the compound LG-H. The activated form may be an activated ester, an activated amide, an anhydride or an acid halide, such as an acyl chloride. Activated esters include derivatives formed by the reaction of a carboxylic acid group with N-hydroxybenzotriazole or N-hydroxysuccinimide.

"activated carbonate" refers to a carbonate comprising a-OC (O) OR moiety, where OR represents a good leaving group capable of coupling the activated carbonate with an amino group to form a carbamate moiety and liberate the compound ROH. The R group of the activated carbonate includes, but is not limited to, p-nitrophenyl, pentafluorophenyl, 2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl, and benzyl, preferably p-nitrophenyl and pentafluorophenyl.

When the linker has the following formula (III):

the drug-linker moiety has the following formula (IV):

and the final step in the synthesis of the drug-linker moiety is typically the coupling of a compound of formula (V) below with a compound of formula (VI) below:

wherein L is₂As defined above, and LG represents a leaving group, in particular a halide such as chloride, or a group derived from N-hydroxysuccinimide,

H-(W)_w-(Y)_y-D (VI)。

when Y ═ 1 and Y ═ PAB-carbonyl, compounds of formula (VI) can be prepared by coupling Drug (DH) with a compound of formula (VII) below, preferably in protected form:

G-(W)_w-PAB-CO-OR (VII)

wherein W and W are as previously defined, R is as defined in the definition of "activated carbonate", and G is H or a protecting group.

When the compound of formula (VII) is in protected form, a final deprotection step is necessary.

When y is 0, compound (VI) has the formula H- (W)_w-D, wherein (W)_wAnd preferably D consists of amino acid units. In this case, therefore, compound (VI) can be prepared by conventional peptide synthesis methods well known to those skilled in the art.

Ab-linker-drugs

A preferred embodiment according to the present invention consists of the conjugation of a cysteine present on the antibody to an electrophilic group of the drug-linker moiety, preferably of a cysteine present on the antibody to a maleimide moiety present on the drug-linker moiety.

The maleimide-cysteine coupling may be performed by methods well known to those skilled in the art.

Typically, antibodies do not contain many, if any, free and reactive cysteine thiol groups that can be attached to a drug moiety. Most cysteine thiol residues in antibodies exist as disulfide bridges and must be reduced with a reducing agent such as Dithiothreitol (DTT) or TCEP under partially or fully reducing conditions. The loading (drug/antibody ratio) of the ADC can be controlled in a number of different ways, including: (i) limiting the molar excess of drug-linker intermediate (D-L) or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reduction conditions for cysteine thiol modification.

the disulfide bond structure of human IgG is now well established (reviewed in Liu and May, mAbs4(2012): 17-23). in fact, for the disulfide bond structures of the 4 human IgG subclasses (i.e. IgG1, IgG2, IgG3 and IgG4), there are many similarities and some differences.

Additional nucleophilic groups can be introduced into the antibody by reaction of lysine with 2-iminothiolane (Traut's) reagent, converting the amine to a thiol. Reactive thiol groups can also be introduced into an antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., making a mutant antibody comprising one or more non-native cysteine amino acid residues). US 7521541 teaches the engineering of antibodies by the introduction of reactive cysteine amino acids.

cysteine amino acids may be engineered at the reactive site of an antibody and they do not form intra-or intermolecular disulfide bonds (Junutula, et al, 2008b Nature biotech, 26(8): 925-932; Dornan et al (2009) Blood114(13): 2721-2729; US 7521541; US 7723485; WO 2009/052249.) the engineered cysteine thiol may be reacted with a linker reagent or drug-linker reagent of the present invention to form an ADC having a cysteine engineered antibody and a drug moiety, the linker reagent or drug-linker reagent having a thiol-reactive electrophilic group such as a maleimide or α -haloamide.

When more than one nucleophilic or electrophilic group of an antibody reacts with a drug-linker intermediate, or with a linker reagent and then with a drug moiety reagent, the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to the antibody, e.g., 1,2,3, etc. Liquid chromatography, such as polymeric reversed phase (PLRP) and Hydrophobic Interaction (HIC), can separate compounds in a mixture by drug loading value. Formulations of ADCs with a single drug loading value (p) can be isolated, however, these single loading values of ADCs are still likely to be a heterogeneous mixture, as the drug moiety can be attached to different sites of the antibody via a linker.

For some antibody-drug conjugates, the drug ratio may be limited by the number of attachment sites on the antibody. High drug loadings (e.g., drug ratios >5) can result in aggregation, insolubilization, toxicity or loss of cell permeability of certain antibody-drug conjugates. Typically, a drug moiety smaller than the theoretical maximum is conjugated to the antibody during the conjugation reaction.

Drug loading, also known as the drug-antibody ratio (DAR), is the average amount of drug per cell binding agent.

For antibodies of the IgG1 and IgG4 isotypes, in which the drug binds to cysteine after partial antibody reduction, the drug loading may be 1 to 8 drugs (D) per antibody, i.e., where 1,2,3, 4, 5,6, 7, and 8 drug moieties are covalently linked to the antibody.

For the antibody IgG2 isotype, where the drug binds to cysteine after partial antibody reduction, the drug loading may be 1 to 12 drugs (D) per antibody, i.e., where 1,2,3, 4, 5,6, 7, 8, 9, 10, 11, and 12 drug moieties are covalently attached to the antibody.

The compositions of ADCs comprise a collection of cell binding agents (e.g., antibodies) conjugated with 1 to 8 or 1 to 12 drugs.

The average number of drugs per antibody in the preparation of ADCs from the conjugation reaction can be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectrometry, ELISA analysis and electrophoresis.

As a non-limiting embodiment, provided herein is conjugation to antibody c208F 2. In this case, the drug is coupled to at least one cysteine selected from i) the residue Cys at position 214 of the light chain of sequence SEQ ID No.28, and ii) the residues Cys at positions 223, 229 and 232 of the heavy chain of sequence SEQ ID No. 23.

As a non-limiting embodiment, provided herein is conjugation to antibody c208F 2. In this case, the drug is conjugated to two, three or four cysteines selected from i) the residue Cys at position 214 of the light chain of sequence SEQ ID No.28, and ii) the residues Cys at positions 223, 229 and 232 of the heavy chain of sequence SEQ ID No. 23.

As a non-limiting embodiment, provided herein is conjugation to the antibody hz208F2 (ar.1). In this case, the drug is coupled to at least one cysteine selected from i) the residue Cys at position 214 of the light chain of sequence SEQ ID No.39, and ii) the residues Cys at positions 223, 229 and 232 of the heavy chain of sequence SEQ ID No. 37.

As a non-limiting embodiment, provided herein is conjugation to the antibody hz208F2 (var.3). In this case, the drug is conjugated to two, three or four cysteines selected from i) the residue Cys at position 214 of the light chain of sequence SEQ ID No.40, and ii) the residues Cys at positions 223, 229 and 232 of the heavy chain of sequence SEQ ID No. 38.

An alternative consists of lysine coupling. The antibody may contain, for example, a number of lysine residues that are not reactive with the drug-linker intermediate (D-L) or linker reagent. Only the most reactive lysine groups can react with the amine-reactive linker reagent. Furthermore, only the most reactive cysteine thiol group can react with the thiol-reactive linker reagent.

When the compound of the invention binds to lysine, the drug loading may be from 1 to 80 drugs (D) per cell antibody, but a preferred upper limit is 40, 20, 10 or 8. The compositions of ADCs comprise a collection of cell binding agents (e.g., antibodies) conjugated with 1 to 80, 1 to 40, 1 to 20, 1 to 10, or 1 to 8 drugs.

The ADC of formula (I) according to the invention may be in the form of a pharmaceutically acceptable salt.

In the present invention, "pharmaceutically acceptable" means that it can be used in the formulation of a pharmaceutical composition, is generally safe, non-toxic, neither biologically nor otherwise undesirable, and is acceptable for veterinary use as well as human pharmaceutical use.

By "pharmaceutically acceptable salt" of a compound is meant a pharmaceutically acceptable salt, as defined herein, and which possesses the desired pharmacological activity of the parent compound.

The pharmaceutically acceptable salts comprise, inter alia:

(1) pharmaceutically acceptable acid addition salts with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and the like; pharmaceutically acceptable acid addition salts with pharmaceutically acceptable organic acids such as acetic acid, trifluoroacetic acid, propionic acid, succinic acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, methanesulfonic acid, stearic acid, lactic acid and the like; and

(2) pharmaceutically acceptable base addition salts formed when the acid proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, alkaline earth metal ion, or aluminum ion; or a pharmaceutically acceptable base addition salt formed when coordinated to a pharmaceutically acceptable organic base, such as lysine, arginine, and the like; or a pharmaceutically acceptable base addition salt formed when coordinated to a pharmaceutically acceptable inorganic base, such as sodium hydroxide, potassium hydroxide (potash), calcium hydroxide, and the like.

These salts can be prepared from the compounds of the invention containing a base or acid functional group with the corresponding acid or base using conventional chemical methods.

V-treatment

Finally, the invention relates to an ADC as described above for use as a medicament, in particular for the treatment of cancer.

Another subject of the invention is a compound of formula (I) as defined above, for its use as a pharmaceutical product, in particular for the treatment of cancer.

The invention also relates to the use of a compound of formula (I) as defined above for the manufacture of a pharmaceutical product, in particular intended for the treatment of cancer.

The present invention also relates to a method for the treatment of cancer comprising administering to a human in need thereof an effective amount of a compound of formula (I) as defined above.

The cancer may preferably be selected from IGF-1R-associated cancers comprising tumor cells expressing or overexpressing all or part of the protein IGF-1R on their surface.

More particularly, the cancer is breast cancer, colon cancer, esophageal cancer, hepatocellular cancer, gastric cancer, glioma, lung cancer, melanoma, osteosarcoma, ovarian cancer, prostate cancer, rhabdomyosarcoma, kidney cancer, thyroid cancer, endometrial cancer, schwannoma, neuroblastoma, oral squamous cell carcinoma, mesothelioma, leiomyosarcoma, and any sign or cancer of resistance to drugs.

For the avoidance of doubt, it must be understood that a drug-resistant IGF-1R-expressing cancer refers not only to a drug-resistant cancer that initially expresses IGF-1R, but also to a cancer that does not initially express or overexpress IGF-1R, but which expresses IGF-1R when it has become resistant to previous treatment.

Another object of the invention is a pharmaceutical composition comprising an ADC as described in the specification.

More particularly, the present invention relates to a pharmaceutical composition comprising an ADC of the present invention and at least one excipient and/or pharmaceutically acceptable carrier.

In the present specification, the expression "pharmaceutically acceptable carrier" or "excipient" is intended to mean a compound or a combination of compounds which enters into a pharmaceutical composition without causing secondary reactions and which allows, for example, a simplification of the administration of the active compound, an increase in the longevity and/or efficacy of the active compound in vivo, an increase in the solubility of the active compound in solution or an improvement in the preservation of the active compound. Such pharmaceutically acceptable carriers and excipients are well known to those skilled in the art and will vary depending on the nature of the active compound selected and the mode of administration.

The active ingredients can be administered to animals or humans in unit administration form, in admixture with conventional pharmaceutical carriers. Suitable unit administration forms include forms via the oral route, and forms via the parenteral route (subcutaneous, intradermal, intramuscular or intravenous).

As solid compositions for oral administration, tablets, pills, powders (hard or soft gelatin capsules) or granules may be used. In these compositions, the active ingredient of the invention is mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose or silicon dioxide, in a stream of argon gas. These compositions may also contain substances other than diluents, for example one or more lubricants such as magnesium stearate or talc, colorants, coatings (coated tablets) or varnishes (varnish).

Sterile compositions for parenteral administration may preferably be aqueous or non-aqueous solutions, suspensions or emulsions. As solvents or carriers, water, propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil, injectable organic esters such as ethyl oleate or other suitable organic solvents can be used. These compositions may also contain adjuvants, in particular wetting agents, isotonicity agents, emulsifiers, dispersants and stabilizers. Sterilization can be performed in a variety of ways, such as by sterile filtration, by adding a sterilizing agent to the composition, by radiation, or by heating. They may also be prepared in the form of solid sterile compositions which may be dissolved at the time of use in sterile water or any other injectable sterile medium.

Preferably, these ADCs will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal or subcutaneous route or by the oral route. In a more preferred manner, the composition comprising ADCs according to the invention will be administered in a sequential manner a plurality of times.

The invention therefore also relates to a kit comprising at least i) an antibody-drug-conjugate according to the invention and/or a pharmaceutical composition according to the invention and ii) a syringe or vial or ampoule in which the antibody-drug-conjugate and/or the pharmaceutical composition is contained.

Their mode of administration, dosage and optimal pharmaceutical form can be determined according to criteria usually considered when establishing a treatment suitable for a patient, such as the age or weight of the patient, the severity of his/her overall condition, tolerance to the treatment and noted side effects.

Other features and advantages of the invention appear in the continuation of the description with the embodiments and the figures, the description of which is presented below.

Drawings

FIGS. 1A-1C: binding of the antibody to human native IGF-1R by FACS analysis. FIG. 1A shows a titration curve in the MCF-7 cell line. MFI represents the mean value of fluorescence intensity. FIG. 1B shows the EC of murine anti-IGF-1R antibody and chimeric anti-IGF-1R antibody in MCF-7 cell line₅₀. FIG. 1C shows B of chimeric anti-IGF-1R antibodies in MCF-7 cell line_max。

FIGS. 2A-2B: evaluation of hIGF-1R recognition using transfected versus untransfected cells. FIG. 2A shows a schematic representation of the IGF-1R⁺Titration curves for one chimeric anti-IGF-1R Ab in cell lines. MFI represents the mean value of fluorescence intensity. FIG. 2B shows the expression in human IGF-1R^-Binding of chimeric anti-IGF-1R Ab in cell lines.

FIGS. 3A-3B: evaluation of the specificity of the abs for IGF-1R for hIR using transfected cells. FIG. 3A shows a block diagram at hIR⁺Binding of murine anti-IGF-1R Ab in transfected cell lines. FIG. 3B shows the binding of chimeric anti-IGF-1R Ab in IR + cell lines. MFI represents the mean value of fluorescence intensity. GRO5 anti-hIR Mab (Calbiochem) was introduced as a positive control.

FIG. 4: murine anti-IGF-1R Ab binding in IM-9 cell lines. MFI represents the mean value of fluorescence intensity. GRO5 anti-hr Mab was introduced as a positive control.

FIGS. 5A-5C: evaluation of recognition of monkey IGF-1R. FIG. 5A shows a titration curve of chimeric anti-IGF-1R Ab in COS-7 cell line. MFI represents the mean value of fluorescence intensity. FIG. 5B shows the EC of murine anti-IGF-1R antibody and chimeric anti-IGF-1R antibody in COS-7 cell line₅₀. FIG. 5C shows EC of chimeric anti-IGF-1R antibodies in NIH 3T 3-transfected cell hIGF-1R + and COS-7 cell lines₅₀。

FIG. 6: sensorgrams (sensorgram) obtained on Biacore X100 based on SPR technology using a CM5 sensor chip (sensorhip), said CM5 sensor chip being activated with more 11000RU of mouse anti-Tag His antibody, which was chemically grafted onto a carboxymethyl dextran matrix. The experiment was run at 25 ℃ at a flow rate of 30. mu.l/min using HBS-EP + as run and sample dilution buffer. The figure shows the overlay of 4 independent sensorgrams aligned on the x-axis at the beginning of the first injection of analyte, and the overlay aligned on the y-axis by the baseline defined immediately before this first injection. Sensorgrams obtained by capturing the sequence of human-based recombinant soluble IGF1R were labeled with diamonds. Sensorgrams obtained by capturing the sequence of cynomolgus monkey-based recombinant soluble IGF-1R were labeled with triangles. White symbols correspond to blank cycles (5 injections of running buffer) and black symbols correspond to injections of c208F2(5, 10, 20, 40, and 80nM) at increasing concentration ranges.

FIG. 7: evaluation of the intrinsic effect of anti-hogf-1R antibodies on receptor phosphorylation compared to IGF 1.

FIG. 8: inhibition of IGF-1R phosphorylation in IGF-1 by murine anti-hIGF-1R.

FIG. 9: after incubation of the cells at 37 ℃, the cell surface signal intensity of the anti-IGF-1R antibody was down-regulated. MCF-7 cells were incubated with 10. mu.g/ml Ab for 4h at 4 ℃ or 37 ℃. The diagram shows Δ MFI.

FIGS. 10A-10B: antibody surface attenuation (decay). Cell surface bound antibodies were assessed after 10, 20, 30, 60 and 120 minutes at 37 ℃. FIG. 10A shows the% of IGF-1R remaining compared to the signal intensity measured at 4 ℃. Figure 10B represents half-life calculations using Prism software and using exponential decay fitting.

FIG. 11: anti-hIGF-1R Ab is internalized. Cells were incubated with 10. mu.g/ml murine Ab at 37 ℃ for 0, 30 or 60 min. Cells were permeabilized (permeabilize) or not and incubated with a second anti-mouse IgG-Alexa 488. The membrane corresponds to signal intensity w/o permeabilization (permeabilization). The sum corresponds to the signal intensity after cell permeabilization, and the cytoplasm corresponds to the internalized Ab. The name of each antibody evaluated is noted at the top of each graph.

FIGS. 12A-12B: imaging of Ab internalization. FIG. 12A: MCF-7 cells incubated with m208F2 for 20 minutes at 4 ℃ and washed prior to incubation (W), incubated at 37 ℃ for 15min (X), 30min (Y) and 60min (Z). Cells were fixed and permeabilized. M208F2 Ab was shown using anti-mouse IgG Alexa488 and Lamp-1, and rabbit anti-Lamp-1 antibody and a second anti-rabbit IgG Alexa 555. FIG. 12B: MCF-7 cells were incubated with anti-hIGF-1R murine antibody for 30 minutes at 37 ℃ and stained as described above. Co-location was determined using the co-location highlight (highlieter) plug-in of ImageJ software.

FIG. 13: involvement of the lysosomal pathway in antibody degradation.

FIG. 14: acidic pH decreased the binding capacity of five murine anti-IGF-1R antibodies.

FIGS. 15A-15D: binding characteristics of the first humanized form of c208F2 Mab. The binding properties of hz208F2 VH3/VL3mAb were evaluated in human cell line MCF-7(A), monkey cell line COS-7(B) and transfected murine cell line expressing human insulin receptor (C). Binding of murine 208F2 mAb and chimeric 208F2 mAb were evaluated in parallel. GRO5 was cloned using an anti-hIR antibody to verify hIR expression in transfected cell line (D).

FIG. 16: the surface of the hz208F2 VH3/VL3 antibody decayed.

FIG. 17: superposition of sensorgrams obtained with a CM5 sensor chip activated with a Biacore X100 device based on SPR in two flow cells (flowcell) with a mouse anti-TagHis monoclonal antibody at about 12.000RU, chemically grafted onto a carboxymethyl dextran matrix, using HBS-EP + as running buffer, at a flow rate of 30 μ l/min, at a temperature of 25 ℃. Each sensorgram (first marked with triangles and second marked with diamonds) corresponds to one complete cycle:

1-injection of a solution of recombinant h-IGF-1R (10. mu.g/ml) in a second flow cell over a one minute period.

2-for the first sensorgram: 5 injections of running buffer, each for 90s

For the second sensorgram: five injections of anti-IGF-1R c208F2 antibody solutions of increasing concentration range, 90s each.

A delay of 3-300s for determining the dissociation kinetic rate.

4-regeneration of the surface by injection of 10mM glycine, HCl pH 1.5 buffer for 45 s.

FIG. 18: sensorgrams are shown in grey, corresponding to sensorgrams obtained with increasing concentration ranges of anti-IGF-1 Rc208F2 solution minus the blank sensorgram (5 injections of HBS-EP +). The theoretical sensorgram corresponding to the 1:1 model is shown in thin black lines with the following parameters: k is a radical of_on＝(1.206±0.036)x10⁶M^-1.s^-1，k_off＝(7.81±0.18)x10^-5s^-1Rmax is 307.6 + -0.3 RU. The calculated concentration of c208F2 is reported on the graph: only the highest concentration (24nM) was considered constant).

FIG. 19: the dissociation constant corresponds to the average of four experiments run with each antibody and to the ratio expressed in pM units: k is a radical of_off/k_onx 10¹². The error bars correspond to the standard error (n-4).

FIG. 20: the half-life corresponds to the average of four experiments run with each antibody and to the ratio expressed in h units: ln (2)/k_off/3600. The error bars correspond to the standard error (n-4).

FIG. 21: cytotoxicity against IGF-1R conjugated to three different compounds. Five chimeric antibodies anti-IGF-1R were conjugated to E-13, G-13 or F-63. An unrelated antibody, c9G4, was also conjugated to the same compound.

FIGS. 22A-22C: in vivo evaluation of C208F2-E-13 (FIG. 22A), C208F2-G-13 (FIG. 22B) and C208F2-F-63 (FIG. 22C) in the MCF-7 xenograft model.

FIGS. 23A-23B: in vivo evaluation of c208F2-E-13 (FIG. 23A) and c208F2-G-13 (FIG. 23B) compared to ADC controls (c9G4-E13 and c9G4-G-13) in the MCF-7 xenograft model.

Fig. 24A and B: acidic pH reduced the binding capacity of humanized IGF-1R antibodies hz208F 2H 076/L024(A) and hz208F2(H077/L018 (B)). FIG. 25: evaluation of cytotoxicity of c208F2-G-13 on Normal cells.

FIG. 26: cytotoxicity of humanized variants of hz208F2 coupled to G-13. An unrelated antibody, c9G4, was also conjugated to the same compound.

FIG. 27 is a schematic view showing: in vivo evaluation of the humanized form of 208F2-G-13 compared to the humanized form of c208F2-G-13 in the MCF-7 xenograft model.

Fig. 28A and 28B: in the MCF-7 xenograft model, in vivo evaluation of c208F2-G-13(28A) or hz208F2-4-G-13(28B) with 4 injections compared to one injection.

Fig. 29A and 29B: in vivo evaluation of c208F2-E-13(29A) and c208F2-G-13(29B) in the CaOV-3 xenograft model.

Detailed Description

Examples

Example 1: production of murine antibodies against IGF-1R ECD

To generate murine monoclonal antibodies (Mab) directed against the human extracellular domain (ECD) of the human IGF-1 receptor (hIGF-1R), 5 BALB/c mice were treated with 10. mu.g of rhIGF-1R protein (R)&D Systems, catalog number 391-GR) 3 times. Alternatively, 10 μ g of the murine extracellular domain (ECD) (R) of IGF-1R was used in some animals&D Systems, catalog number 6630-GR/Fc) were immunized three additional times. The first immunization was performed in the presence of complete Freund's (Freund) adjuvant (Sigma, St Louis, Md., USA). AddingIncomplete Freund's adjuvant (Sigma) was used for subsequent immunization. Three days prior to fusion, immunized mice were boosted with 10 μ g of rhIGF-1R protein. Splenocytes and lymphocytes were then prepared by perfusion of the spleen, and by mincing (minse) proximal lymph node, respectively, harvested from 1 of 5 immunized mice (selected after titration of all mice' sera) and fused with SP2/0-Ag14 myeloma cells (ATCC, Rockville, MD, usa). Fusion protocols are described by Kohler and Milstein (Nature,256:495-497, 1975). The fused cells are then subjected to HAT selection. In general, for the preparation of monoclonal Antibodies or functional fragments thereof, in particular of murine origin, reference can be made in particular to the techniques described in the Manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, page 726, 1988). Approximately 10 days after fusion, colonies of hybrid cells were screened. For primary screening, hybridoma supernatants were evaluated for secretion of mabs to IGF-1RECD protein by FACS analysis using human breast MCF7 tumor cells (ATCC) and/or monkey COS7 cells (transformed by african green monkey kidney-SV 40) that express monkey IGF-1R on their cell surface. More precisely, for selection by flow cytometry, 10 was added at 4 ℃⁵Individual cells (MCF7 or COS7) were seeded into PBS containing 1% BSA and 0.01% sodium azide (FACS buffer) in each well of a 96-well plate. After centrifugation at 2000rpm for 2min, the buffer was removed and the hybridoma supernatant to be tested was added. After incubation at 4 ℃ for 20min, the cells were washed twice, Alexa 488-conjugated goat anti-mouse antibody 1/500 ° (# a11017, Molecular Probes inc., Eugene, usa) diluted in FACS buffer was added, and incubated at 4 ℃ for 20 min. After final washing with FACS buffer, cells were analyzed by FACS (Facscalibur, Becton-Dickinson) after adding propidium iodide to each tube at a final concentration of 40 μ g/ml. Wells containing cells alone and wells containing cells incubated with secondary Alexa 488-conjugated antibody were included as negative controls. An isotype control (Sigma, reference M90351MG) was used in each experiment. At least 5000 cells were evaluated to calculate the mean value of fluorescence intensity (MFI).

In addition, an internalization assay was performed to select only internalizing antibodies. For this analysis, the MCF7 tumor cell line was cultured for 3 days in RMPI 1640 with 1% L-glutamine and 10% FACS without phenol red before the experiment. Then, cells were dispersed using trypsin, and 100. mu.l of 4.10 cells were added⁵Cell suspensions of individual cells/ml were seeded into 96-well plates in phenol red-free RPMI1640 with 1% L-glutamine and 5% FBS. After centrifugation at 2000rpm for 2min, the cells were resuspended in 50. mu.l of hybridoma supernatant or control antibody solution (positive and isotype control at 1. mu.g/ml). After incubation at 4 ℃ for 20min, the cells were centrifuged at 2000rpm for 2min and resuspended in either cold (4 ℃) or warm (37 ℃) complete culture medium. Then, the cells were incubated at 37 ℃ or at 4 ℃ for 2 hours. Then, the cells were washed three times with FACS buffer. The Alexa 488-labeled goat anti-mouse IgG antibody was incubated for 20 minutes, and the cells were washed three times before FACS analysis in a propidium iodide negative cell population.

After FACS analysis, two parameters were determined: (i) the difference in the fluorescence signal detected on the surface of cells incubated with a hybridoma supernatant at 4 ℃, from the fluorescence signal obtained from cells incubated with a hybridoma supernatant at 37 ℃, and (ii) the percentage of IGF-1R remaining on the cell surface.

The percentage of hIGF 1R remaining was calculated as follows: the remaining IGF-1R% (MFI)_{Ab 37℃}/MFI_{Ab 4℃})x 100。

In addition, three ELISAs (before or after cloning) were performed to study the binding of antibodies to recombinant human (hIGF-1R) and murine (miggf-1R) proteins, and to the recombinant human insulin receptor (hIR) protein. Hybridomas secreting antibodies that show binding to rh-and/or rm-IGF-1R and no binding to rhIR are retained. Briefly, 96-well ELISA plates (Costar 3690, Corning, NY, USA) were coated overnight at 4 ℃ with 100. mu.l/well in PBS with 0.6. mu.g/ml of rhIGF-1R protein (R & D Systems, Cat 391-GR), or 1. mu.g/ml of rmIGF-1R protein (R & D Systems, Cat 6630-GR/Fc), or 1. mu.g/ml of rhIR protein (R & D Systems, Cat 1544-IR/CF). The plates were then blocked with PBS containing 0.5% gelatin (#22151, Serva Electrophoresis GmbH, Heidelberg, Germany) for 2 hours at 37 ℃. After the saturation buffer was discarded by shaking the plates, 100. mu.l of each supernatant dilution was added to each well (undiluted hybridoma supernatant or serial dilutions of supernatants) and incubated at 37 ℃ for 1 h. After three washes, 100. mu.l of horseradish peroxidase conjugated polyclonal goat anti-mouse IgG (#115-035-164, Jackson Immuno-research laboratories, Inc., West Grove, Pa., USA) diluted 1/5000 in PBS containing 0.1% gelatin and 0.05% Tween 20(w: w) was added for 1 hour at 37 ℃. Then, the ELISA plate was washed 3 times, and TMB ((# UP664782, Uptima, Interchim, France) substrate was added, after incubation at room temperature for 10min, the reaction was terminated with 1M sulfuric acid, and the optical density at 450nm was measured.

Hybridomas secreting the antibody of interest are amplified and cloned by limiting dilution. Once isotypized, each encoded clone was amplified and frozen. Each antibody of interest was produced in an in vitro production system called CellLine (Integra Biosciences) for further characterization.

Additional analysis of treatment (address) in combination with specific FACS analysis was performed in IM9 cells (B lymphoblastoid cells expressing human IR), and in hIGF-1R transfected cells compared to untransfected cells.

All data corresponding to the selected antibodies are summarized in table 7 and demonstrate that the five selected antibodies strongly recognize native human IGF-1R expressed on MCF-7 breast cancer cells or transfected cells. They also recognized monkey IGF-1R on COS-7 cells. These antibodies do not cross-react with human insulin receptor highly expressed on IM9 cells. It must be noted that these antibodies poorly recognized rhIGF-1R ECD protein when coated directly on ELISA plates.

Example 2: antibody binding to human native IGF-1R by FACS analysis

5 murine IGF-1R antibodies were chimeric. The binding properties of murine IGF-1R antibody and chimeric IGF-1R antibody were evaluated by FACS analysis in human MCF-7 breast cancer cell line (ATCC # HTB-22) using increasing antibody concentrations. For this purpose, in FACS buffer (PBS, 0.1% BSA, 0.01% NaN)₃) In (1X 10), cells were cultured⁶Individual cells/ml) was incubated with IGF-1R antibody for 20min at 4 ℃. They were then washed 3 times and incubated with the appropriate secondary antibody conjugated to Alexa488 at 4 ℃ for an additional 20 minutes in the dark, followed by 3 washes in FACS buffer. Propidium iodide (which stains dead cells) is used to identify viable cells in which binding of anti-IGF-1R antibodies is immediately performed. The maximum value of the signal intensity obtained by each antibody was designed as B_maxAnd expressed as the mean value of fluorescence intensity (MFI). Bound EC was calculated using non-linear regression analysis (GraphPad Prims 4.0)₅₀Expressed as molar concentration (M).

Titration curves for each murine or chimeric Ab demonstrated that all antibodies produced were able to recognize the native IGF-1R form in a typical saturation pattern (profile) (fig. 1A). To rank the antibodies and compare the binding properties of murine abs and chimeric abs, binding EC of each compound was determined using non-linear regression analysis₅₀. EC for each murine Ab and its corresponding chimeric form₅₀Comparison of (A) shows that 2 forms show the same binding properties, indicating that Ab chimerism does not affect IGF-1R recognition (FIGS. 1B-C). EC of chimeric antibody₅₀And B_maxThe values are summarized in table 8.

TABLE 8

AC	B_max	EC₅₀
			c208F2	981	6.7E-10
c212A11	991	6.7E-10
			c214F8	1069	5.0E-10
c219D6	993	4.7E-10
			c213B10	1103	4.4E-10

Example 3: confirmation of antibody specificity by cells transfected with IGF-1R or IR or IM9 cells IM9 cells express significant levels of IR

To confirm the specificity of the generated antibodies for IGF-1R compared to IR, stable transfectants expressing hIGF-1R or hIR were evaluated by FACS analysis. Briefly, in FACS buffer (PBS, 0.1% BSA, 0.01% NaN)₃) In (3), increasing concentrations of chimeric mAb and cells at 4 degrees C were incubated for 20 min. The cells were then washed 3 times and incubated with a suitable secondary antibody conjugated to Alexa488,then incubated in the dark at 4 ℃ for another 20 minutes, then washed 3 times in FACS buffer. Propidium iodide (which stains dead cells) is used to identify viable cells in which binding of anti-IGF-1R antibodies is immediately performed. Bound EC was calculated using non-linear regression analysis (GraphPad Prims 4.0)₅₀Expressed as molar concentration (M).

Binding specificity of the chimeric Ab to human IGF-1R was confirmed in hIGH-1R transfected cell lines (FIG. 2A) compared to titration curves obtained in untransfected cells (FIG. 2B). EC (EC)₅₀And B_maxThe values are summarized in table 9.

TABLE 9

Ac	B_max	EC₅₀(M)
			c208F2	2008	3.2E-10
c212A11	2513	4.4E-10
			c214F8	2094	2.7E-10
c219D6	2521	5.5E-10
			c213B10	2029	3.3E-10

To confirm the absence of binding of murine and chimeric antibodies to hIR, a stable cell line expressing human IR (hIR) was used. The recognition of murine abs and chimeric abs to human cell surface hIR was performed by FACS analysis. In FACS buffer (PBS, 0.1% BSA, 0.01% NaN)₃) In (b), increasing concentrations of a murine mAb or a chimeric mAb are added at hIR⁺Transfected cell lines were incubated at 4 ℃ for 20 minutes. The cells were then washed 3 times and incubated with the appropriate secondary antibody conjugated to Alexa488, followed by incubation in the dark at 4 ℃ for an additional 20 minutes, followed by 3 washes in FACS buffer. Propidium iodide (which stains dead cells) is used to identify viable cells in which binding of anti-IGF-1R antibodies is immediately performed. Bound EC was calculated using non-linear regression analysis (GraphPad Prims 4.0)₅₀Expressed as molar concentration (M). The anti-hIR antibody clone GRO5 was used as a positive control. The murine 9G4 antibody and the chimeric 9G4 antibody were introduced as unrelated antibodies.

High level expression of hIR on the cell surface of transfected cells was confirmed using commercially available anti-hIR antibody GRO5 (fig. 3A and 3B). No observation was made at hIR even with high concentrations of murine hIGF-1R Ab (FIG. 3A) or chimeric hIGF-1R Ab (FIG. 3B)⁺Binding on the cell surface of transfected cells. These results demonstrate that neither murine anti-hIGF-1R Ab nor chimeric anti-hIGF-1R Ab recognized hIR.

The specificity of recognition of this hIGF-1R compared to IR was also demonstrated by FACS analysis using IM9 cells (B lymphoma cell line expressing hIR) (FIG. 4). For this FACS analysis, the protocol was the same as described above, and a murine antibody was used to prevent cross-reactivity of the secondary anti-human Ab (IM9 cells express human Ig on their cell surface). The results presented in figure 4 again demonstrate that the expected signal was observed using the GRO5 anti-hIR antibody, whereas none of the murine antibodies evaluated showed any significant binding signal in this cell line.

Example 4: antibody binding to monkey native IGF-1R by FACS and Biacore analysis

One of the first prerequisites for regulatory toxicology studies is to find relevant animal species for evaluation of selected compounds. Since the panel of antibodies described herein do not recognize murine IGF-1R, the most likely species for toxicological evaluation is non-human primate (NHP).

To evaluate the binding of anti-IGF-1R antibodies to monkey IGF-1R, binding of murine anti-hIGF-1R antibody and chimeric anti-hIGF-1R antibody was first evaluated in a COS-7 cell line by FACS analysis using increasing antibody concentrations. In FACS buffer (PBS, 0.1% BSA, 0.01% NaN)₃) In (1X 10), cells were cultured⁶Individual cells/ml) was incubated with anti-IGF-1R antibody for 20min at 4 ℃. The cells were then washed 3 times and incubated with the appropriate secondary antibody conjugated to Alexa488, followed by incubation at 4 ℃ for an additional 20 minutes in the dark, and finally washed 3 times in FACS buffer. Propidium iodide (which stains dead cells) was used to identify viable cells in which binding of anti-IGF-1R antibodies was immediately assessed. Bound EC was calculated using non-linear regression analysis (GraphPad Prims 4.0)₅₀Expressed as molar concentration (M).

Titration curves obtained in COS-7 monkey cell lines showed that all anti-hIGF-1R Abs specifically recognized IGF-1R expressed on the surface of monkey cell lines (FIG. 5A). EC for each of murine Ab and chimeric Ab₅₀The 2 forms showed a better agreement in their binding properties to monkey IGF-1R (fig. 5B). These results indicate that all of the anti-hIGF-1R produced recognized monkey IGF-1R.

Binding of EC to COS-7 cells compared to transfected IGF-1R cells was performed₅₀To verify a chimeric antibody against human IGF-1R compared to monkey IGF-1RMagnitude of body recognition. The results shown in FIG. 5C demonstrate similar recognition of human IGF-1R and monkey IGF-1R by all antibodies.

To confirm recognition of another type of monkey, cells were transfected with cynomolgus monkey of the IGF-1R form to generate soluble monkey IGF-1R ECD, and Biacore experiments were performed with one of the chimeric antibodies (c208F2) to compare its binding properties to hIGF-1R or cynomolgus monkey IGF-1R.

Identification experiments were run on a Biacore X100 device using a CM5 sensor chip activated by anti-Tag His antibody (His capture kit GE Healthcare catalog No. 28-9950-56). Antibodies in excess of 11000RU were chemically grafted onto carboxymethyl dextran matrix using amine kit chemistry. Experiments were performed at 25 ℃ using HBS-EP buffer (GE Healthcare) as the run and sample dilution buffer at a flow rate of 30. mu.l/min. Using a single cycle kinetic protocol, kinetic parameters for binding of the chimeric form of the 208F2 antibody (c208F2) to hoigf-1R compared to cynomolgus (Macaca) IGF-1R were defined.

a solution of soluble recombinant form of IGF-1R heterotetramer consisting of extracellular domains of 2 α and 2 β chains, expressing an additional C-terminal 10-His tag, based on human sequences (R & D Systems catalog No. 305-GR-50) or sequences of one of the cynomolgus monkeys (manufactured in house) was injected in a second flowcell at a dilution defined to capture approximately 160RU antigen for 1 minute, after the capture phase, running buffer was injected 5 times (90 seconds per injection) or for 5 increasing concentration ranges of C208F2 (90 seconds per injection) in both flowcells after the capture phase, at the end of the fifth injection, running buffer was passed over (pass) to define the dissociation rate.

The surface was then regenerated by injection of 10mM glycine, HCl pH 1.5 buffer during 30 s.

The calculated signal corresponds to the difference between the response of flow cell 2 (with captured IGF-1R) and the response of flow cell 1 (without any IGF-1R molecules) (FIG. 6).

For each IGF-1R molecule (human or cynomolgus monkey), the signal due to the injection of c208F2 in the increased concentration range was corrected by subtracting the signal obtained from 5 injections of buffer (double reference). The resulting sensorgrams were analyzed using Biaevaluation software with a 1:1 model. Kinetic rates were assessed either independently (2 kinetic rates of binding of c208F2 to each IGF-1R) or collectively (same kinetic rates of binding of c208F2 to human IGF-1R and cynomolgus monkey IGF-1R). The quality of the fit was assessed by Chi2/Rmax ratio below 0.05 RU.

The kinetic rates of binding (see table 10) defined for each IGF-1R, respectively, were close and the fit of two sensorgrams with the same kinetic rates was of good quality.

The c208F2 antibody similarly recognized recombinant human IGF-1R and cynomolgus monkey IGF-1R with a dissociation constant (KD) of about 0.2 nM. The affinities defined in this study correspond to the functional affinities (avidity) of antibodies at the level of captured human IGF-1R and cynomolgus IGF-1R of about 160 RU.

Watch 10

IGF1R	kon[1/M.s]	koff[1/s]	Kd[nM]	Chi2/Rmax
					Human being	1.52E+06	3.40E-04	0.23	0.045
Macaca fascicularis	1.85E+06	3.10E-04	0.17	0.032
					Human and cynomolgus monkey	1.52E+06	3.33E-04	0.22	0.039

Example 5: intrinsic effects of the generated antibodies on IGF-1R phosphorylation

It is well known that antibodies can induce agonistic (agonistic) effects when bound to tyrosine kinase receptors. Since we do not wish to select such agonist antibodies, the evaluation of hIGF-1R phosphorylation was investigated using chimeric antibodies.

For this purpose, MCF-7 cells were incubated overnight in serum-free medium. Then, IGF-1(100nM) or the Ab to be tested was added (10. mu.g/ml) at 37 ℃ for 10 minutes. The medium was discarded and the cells were scraped (scrape) at 4 ℃ for 90 minutes in lysis buffer (pH 7.5) containing 10mM Tris HCl buffer (pH 7.5), 15% NaCl (1M), 10% detergent cocktail (10mM Tris-HCl, 10% Igepal lysis buffer) (Sigma Chemical Co.), 5% sodium deoxycholate (Sigma Chemical Co.), 1 protease inhibitor cocktail (cocktail) complete TM disc (Roche), 1% phosphatase inhibitor cocktail set II (Calbiochem). Lysates were clarified by centrifugation at 4 deg.C, heated at 100 deg.C for 5min, and maintained at-20 deg.C, or loaded directly onto 4-12% SDS-PAGE gels. The primary antibody was incubated at room temperature for 2hr, then at room temperature for 1hr with HRP-linked secondary antibody. Membranes were washed in TBST before protein visualization with ECL. Blots were quantified using Image J software. The phospho- (phospho-) protein values were normalized with GAPDH. Phosphorylation of hIGF-1R in response to IGF-1 is considered to be 100% stimulation. The effect of anti-hIGF-1R Ab on hIGF-1R phosphorylation was determined as% of phosphorylation induced by IGF-1.

The results depicted in FIG. 7 represent the mean +/-S.D. of% of 3 independent experiments of pIGF-1R in response to chimeric anti-IGF-1R Ab compared to IGF-1. As shown, no significant or minimal (< 10%) phosphorylation of hIGF-1R was detected when MCF-7 cells were incubated with 10 μ g of anti-IGF-1R Ab.

Example 6: inhibition of IGF-1R phosphorylation in response to IGF-1 by murine IGF-1R antibodies

To characterize the selected antibodies, their ability to inhibit IGF 1-induced phosphorylation was investigated. For this purpose, MCF-7 cells were incubated overnight in serum-free medium. Cells were then incubated with murine anti-hIGF-1R Ab for 5 minutes, followed by the addition of IGF-1 for 2 minutes at 37 ℃. The medium was discarded and the cells were scraped for 90 minutes at 4 ℃ in lysis buffer (pH 7.5) containing 10mM Tris HCl buffer (pH 7.5), 15% NaCl (1M), 10% detergent mix (10mM Tris-HCl, 10% Igepal lysis buffer) (Sigma Chemical Co.), 5% sodium deoxycholate (Sigma Chemical Co.), 1 protease inhibitor mix complete TM disc (Roche), 1% phosphatase inhibitor mix set II (Calbiochem). Lysates were clarified by centrifugation at 4 deg.C, heated at 100 deg.C for 5min, and maintained at-20 deg.C, or loaded directly onto 4-12% SDS-PAGE gels. The primary antibody was incubated at room temperature for 2h, then at room temperature for 1hr with HRP-linked secondary antibody. Membranes were washed in TBST before protein visualization with ECL. Blots were quantified using Image J software. Phospho-protein values were normalized with GAPDH. Phosphorylation of hIGF-1R in response to IGF-1 is considered to be 100% stimulation. The effect of anti-hIGF-1R Ab on hIGF-1R phosphorylation was determined as% of phosphorylation induced by IGF-1.

All anti-IGF-1R Ab strongly inhibited hIGF-1R phosphorylation in response to IGF-1 (> 80% reduction) (FIG. 8). The best inhibitors of IGF 1-induced phosphorylation of hIGF-1R are m208F2, m212a11 and m214F8 Mab.

Example 7: study of IGF-1R internalization upon binding of the produced IGF-1R antibody by FACS analysis

MCF-7 cells were incubated with 10. mu.g/ml chimeric antibody at 4 ℃ for 20 min. Then, the cells were washed and incubated at 4 ℃ or 37 ℃ for 4 h. The amount of cell surface bound antibody was determined using a secondary antibody. Δ MFI corresponds to the amount of internalized Ab, defined as the difference between the MFI measured at 4 ℃ and the MFI measured at 37 ℃ after 4 hours incubation time. Δ MFI is presented in fig. 9 and table 11. The percent internalization of 10 μ g/ml Ab was calculated as follows: 100 x (MFI of 4 ℃ to MFI of 37 ℃) to MFI of 4 ℃ and is presented in Table 11.

TABLE 11

Ab	Internalization percent	ΔMFI	ΔMFI_EC₅₀
				c208F2	83	288	1.8E-10
c212A11	80	322	2.7E-10
				c214F8	87	403	2.2E-10
c219D6	80	353	4.4E-10
				c231B10	85	369	2.3E-10

To determine whether an antibody (which also recognizes monkey IGF-1R) is able to internalize the receptor, the same internalization experiment was performed. The results summarized in table 12 demonstrate that all antibodies tested were able to mediate monkey IGF-1R internalization.

TABLE 12

The kinetics of the reduction of cell surface bound antibodies were further evaluated. For this purpose, MCF-7 cells were seeded in 96-well plates and incubated with 10. mu.g/ml mice for 20min at 4 ℃. The cells were then washed to remove unbound antibody and incubated in medium at 37 ℃ for 10, 20, 30, 60 or 120 min. At each time point, cells were centrifuged and then surface labeled with a second anti-mouse IgG-Alexa488 on ice to determine the amount of antibody remaining on the cell surface. The fluorescence intensity of each murine Ab and each time point was normalized by the signal at 4 ℃ (% of IGF-1R remaining) and fitted to an exponential decay to determine the half-life (t 1/2). t1/2 is considered to be the time required to obtain a 50% signal reduction. As illustrated in fig. 10, the surface levels of all murine abs decreased rapidly in the first 30min, and the decrease was almost maximal after 60min of incubation (fig. 10A). The calculated half-life according to murine Ab was between 10 and 18min (fig. 10B).

To verify that the decrease in cell surface signal was due to Ab internalization rather than receptor shedding, cells were incubated with murine abs at 37 ℃ for 0, 30, and 60min (fig. 11). Cells were then fixed and either permeabilized or not permeabilized to determine cell surface bound antibody (w/o permeabilization) and total antibody signal corresponding to cell surface bound + internalized Ab (permeabilized). The amount of internalized Ab (cytoplasm) was determined as follows: permeabilized MFI-w/o permeabilized MFI. This experiment shows that the decrease in cell surface bound abs is due to an increase in cytoplasmic abs, demonstrating that the abs are internalized (fig. 11). In addition, degradation of Ab started after 1h of incubation as shown by the decrease in signal (total) after permeabilization.

Example 8: study of IGF-1R internalization upon binding of produced IGF-1R antibody by confocal analysis

To further confirm antibody internalization, confocal microscopy was performed to assess the subcellular distribution of the antibody following cell trafficking (cellularization). Cells were incubated with anti-hIGF-1R Ab at 37 ℃, fixed and permeabilized. Thus, cells were stained with the secondary antibody Alexa-488 and rabbit anti-Lamp-1 antibody, which was displayed using the secondary anti-rabbit IgG Alexa 555. Murine 208F2 Ab was localized to the membrane of MCF-7 cells prior to incubation at 37 deg.C (FIG. 12A). No co-localization with the lysosomal marker lamp-1 was noted using the co-localization highlight insert of Image J software. After incubation at 37 ℃ for 15min, cell surface bound antibodies were significantly reduced. Intracellular antibodies were detected in vesicles with a decrease in cell surface bound antibodies. A rare co-localization with lamp-1 was observed. After 30min incubation, almost no cell surface bound antibody was detected. However, co-localization of abs into lysosomes was increased. After 1h incubation, intracellular Ab staining decreased and the number of co-localizations with lamp-1 also decreased. The kinetics of this cell surface bound antibody and its intracellular accumulation correlate with the kinetics of antibody surface decay measured by FACS. Furthermore, degradation of murine abs by confocal microscopy started after 1h of incubation, as already described with FACS studies.

All other hIGF-1R murine antibodies were also evaluated for internalization and their co-localization with Lamp-1 (FIG. 12B). After incubation at 37 ℃ for 30min, intracellular antibodies were detected and co-localization with lamp-1 could be observed, indicating that all selected anti-IGF-1R antibodies were efficiently internalized into lysosomes.

Example 9: inhibition of Ab degradation using the lysosomal inhibitor baveromycin a1(Bafilomycin a1)

To confirm that the antibodies reached the lysosomes, where they were degraded, the cells were treated with or without baveromycin a1, a potent inhibitor of lysosomal function. Then, cells were incubated with 10 μ g/ml of the Ab to be tested at 4 ℃, washed and incubated for 2h at 37 ℃. After cell permeabilization, internalized abs were detected using a second anti-mouse IgG-Alexa488 Ab. The addition of baverromycin a1 prevented degradation of the Ab within the cell (fig. 13), indicating that the Ab is efficiently internalized and degraded in lysosomes.

Example 10: effect of pH on antibody-IGF-1R binding

Since antibodies are selected based on their internalization potential and shown above to co-localize with early endosomes prior to entry into the lysosomal compartment, an interesting approach is to select antibodies in which the stability of Ab/hIGF-1R binding of the antibody is adjusted according to the pH environment and preferably antibodies that preferentially dissociate from IGF-1R when the pH environment becomes acidic. Indeed, the main difference between early endosomes and lysosomes is their luminal pH: the pH is about 6 in the endosomal compartment and about 4.5 in the lysosomal compartment.

It is well known that once internalized following ligand binding (IGF1), hIGF-1R returns to the cell surface via a recycling pathway.

Without being bound by theory, the hypothesis described herein is that antibodies that would likely facilitate target recovery to the membrane would be more prone to premature release from their target at acidic pH and therefore may be considered better candidates for ADC methods.

To investigate whether some of our antibodies show such properties and correlate them with cytotoxic activity, murine anti-hIGF-1R Mab binding in MCF-7 cell line was performed in buffers of different pH. Increasing concentrations of murine mAb were incubated in MCF-7 cell line at 4 ℃ for 20min at different pH's ranging from 5 to 8. The cells were then washed 3 times and incubated in FACS buffer with an appropriate secondary antibody conjugated to Alexa 488. Cells were incubated at 4 ℃ for an additional 20 minutes in the dark and then washed 3 times in FACS buffer. Propidium iodide (which stains dead cells) was used to identify viable cells in which binding of anti-hIGF-1R antibodies was immediately performed. Bound EC was calculated using non-linear regression analysis (GraphPad Prims 4.0)₅₀Expressed as molar concentration (M). As illustrated in fig. 14, all of the selected murine anti-IGF-1R antibodies showed lower binding capacity at acidic pH.

Binding of humanized anti-IGF-1R Mab in MCF-7 cell line was performed in buffers of different pH. Increasing concentrations of humanized mAb were incubated in MCF-7 cell line at 4 ℃ for 20min at varying pH ranging from 5 to 8. The cells were then washed 3 times and incubated in FACS buffer with an appropriate secondary antibody conjugated to Alexa 488. Cells were incubated at 4 ℃ for an additional 20 minutes in the dark and then washed 3 times in FACS buffer. Identification of survival Using propidium iodide, which stains dead cellsImmediately performing binding of the anti-IGF-1R humanized antibody in said surviving cells. Bound EC was calculated using non-linear regression analysis (GraphPad Prims 4.0)₅₀Expressed as molar concentration (M). As illustrated in fig. 24, the humanized anti-IGFR antibody showed lower binding ability at acidic pH.

Example 12: evaluation of humanized forms of 208F2 Mab

12.1 evaluation of binding and internalization of the first humanized form hz208F2 VH3/VL3 (also known as hz208F 2H 026/L024)

In MCF-7, COS-7 and NIH 3T3IR⁺The binding of the first humanized form of c208F2 mAb was evaluated in cell lines. Increasing concentrations of m208F2, c208F2 or hz208F2 VH3VL3 were added to each cell line for 20min at 4 ℃. Then, the cells were washed and the binding of the tested mAb was shown using the corresponding secondary antibody. To verify the expression of human IR in transfected cell lines, GRO5 was cloned using a commercially available anti-hIR antibody, the recognition pattern of which is exemplified in (fig. 15D).

Comparison of the humanized form with murine or chimeric forms in MCF-7 (FIG. 15A) cells or monkey COS-7 (FIG. 15B) cells showed a compact pattern of the 3 forms tested. The humanization process did not alter the specificity of antibody recognition, which was fully comparable to murine and chimeric versions in terms of the absence of cross-reactivity on human insulin receptor (fig. 15C).

Calculated EC for the first humanized form of 208F2 in human cell line MCF-7 and monkey cell line COS-7₅₀With EC determined with murine or chimeric versions of mAb 208F2₅₀Similarly.

The ability of mAb hz208F2 VH3/VL3 to be internalized was assessed by flow cytometry. MCF-7 cells were incubated with 10. mu.g/ml antibody at 4 ℃ for 20 min. Then, the cells were washed and incubated at 4 ℃ or 37 ℃ for 4 h. The amount of cell surface bound antibody was determined using a secondary antibody. Δ MFI corresponds to the amount of internalized Ab, defined as the difference between the MFI measured at 4 ℃ and the MFI measured at 37 ℃ after 4 hours incubation time. Δ MFI is presented in fig. 16 and table 13. The percent internalization of 10 μ g/ml Ab was calculated as follows: 100 x (MFI of 4 ℃ to MFI of 37 ℃) to MFI of 4 ℃ and is presented in Table 13. Thus, humanized hz208F2 VH3/VL3 has binding and internalization properties similar to those measured with the corresponding murine 208F2 and chimeric 208F2 antibodies.

TABLE 13a

12.2 subsequent evaluation of the binding of the humanized form of hz208F2

mAb 208F2 was humanized and binding properties of sixteen humanized variants (including the first form described in 12.1) were evaluated. The binding properties of the humanized variants were evaluated by FACS analysis in the human MCF-7 breast cancer cell line and the monkey cell line Cos-7 using increasing antibody concentrations. For this purpose, cells (1X 10) were plated in FACS buffer (PBS, 0.1% BSA, 0.01% NaN3)⁶Individual cells/ml) was incubated with anti-IGF-1R antibody for 20min at 4 ℃. They were then washed 3 times and incubated with the appropriate secondary antibody conjugated to Alexa488 at 4 ℃ for an additional 20 minutes in the dark, followed by 3 washes in FACS buffer. Propidium iodide (which stains dead cells) is used to identify viable cells in which binding of anti-IGF-1R antibodies is immediately performed. Bound EC was calculated using non-linear regression analysis (GraphPad Prims 4.0)₅₀Expressed as molar concentration (M).

EC of humanized variants₅₀It was shown that all humanized variants showed equivalent binding properties in human and monkey cell lines.

EC of humanized antibody₅₀Summarized in table 13 b.

TABLE 13b

12.3 evaluation of internalization of another hz208F2 humanized form

MCF-7 cells were incubated with 10. mu.g/ml of humanized antibody at 4 ℃ for 20 min. Then, the cells were washed and incubated at 4 ℃ or 37 ℃ for 4 h. The amount of cell surface bound antibody was determined on a FacsCalibur flow cytometer (Becton Dickinson) using a secondary antibody. Δ MFI corresponds to the amount of internalized Ab, defined as the difference between the MFI measured at 4 ℃ and the MFI measured at 37 ℃ after 4 hours incubation time. Δ MFI is presented in table 13 c. The percent internalization of 10 μ g/ml Ab was calculated as follows: 100 x (MFI at 4 ℃ to MFI at 37 ℃) to MFI at 4 ℃. The humanized antibody hz208F 2H 077/L018 was able to induce significant internalization of IGF-1R.

TABLE 13c

	ΔMFI	Internalization percent
			hz208F2 H077/L018	468	88

Example 13: five chimeric anti-IGF-1R antibodies (c208F2, c213B10, c212A11, c214F8 and c219D6) _DAnd a humanized form of the 208F2 antibody (VH3 `)VL3) dissociation constant (K) for binding in soluble recombinant human IGF-1R Definition of

By dissociation rate (k)_off) And the rate of binding (k)_on) The ratio between defines the dissociation constant (K) for binding of the antibody in recombinant soluble human IGF-1R_D). Kinetic experiments were run in a Biacore X100 apparatus using a CM5 sensor chip activated by a mouse anti-Tag His monoclonal antibody. Using amine kit chemistry, about 12000RU of antibody was chemically grafted onto the carboxymethyl dextran matrix.

Experiments were performed at 25 ℃ using HBS-EP + buffer (GE Healthcare) as the run and sample dilution buffer at a flow rate of 30. mu.l/min.

A single cycle kinetic protocol was used to define the kinetic parameters for the binding of anti-IGF-1R antibodies captured by their two C-terminal 10 histidine tags in soluble recombinant human IGF-1R.

solution of soluble recombinant form of 1-human IGF-1R heterotetramer extracellular domains expressing the 2 α and 2 β chains of an additional C-terminal 10-His tag (R & DSystems catalog No. 305-GR-50) were injected in a second flow cell at a concentration of 10 μ g/ml over a one minute period capturing an average 587RU (with standard deviation of 24RU) of soluble receptor in each of the 24 cycles achieved in this study.

2-after the capture phase, in both flow cells, the running buffer was injected 5 times (90 seconds per injection), or one of the six antibodies was injected in increasing 5 concentration ranges (90 seconds per injection). At the end of the fifth injection, the running buffer was passed over a 5 minute period to define the off-rate.

3-then, the surface was generated by injection of 10mM glycine, HCl pH 1.5 buffer during 45 s.

The calculated signal corresponds to the difference between the response of flow cell 2 (with captured IGF-1R) and the response of flow cell 1 (without any IGF-1R molecules).

The signals due to the injection of one antibody at increasing concentration ranges were corrected for each IGF-1R by subtracting the signals obtained from 5 injections of buffer (double reference), see fig. 17.

The resulting sensorgrams were analyzed by Biaevaluation software with a 1:1 model.

For each antibody, four experiments were run using two different concentration ranges: for each antibody run, the first two experiments were 40, 20, 10, 5 and 2.5nM, the last two experiments were 24, 12, 6, 3 and 1.5 nM.

For the 6 antibodies tested in this experiment, the experimental data and the results with significant k were obtained when the higher concentration was defined as a constant and the other four concentrations were calculated_offThe 1:1 model of values fits well (see fig. 18).

As a ratio k_off/k_onCalculated dissociation constant (K)_D) And as the ratio Ln (2)/k_offThe calculated half-lives of the complexes are presented in fig. 19 and 20. They correspond to the average of four independent experiments run for each antibody. The error bars correspond to the standard error of the numerical values (n-4).

The dissociation constant is in the range of 10 to 100 pM. The c208F2 antibody exhibited a weaker affinity (higher dissociation constant value) for h-IGF-1R (with a K of about 75pM_D) And its humanized form is at least as good as the chimeric form (with a K of about 60 pM)_D). The other four anti-IGF-1R chimeric antibodies showed very similar affinities for hIGF1-R (K)_DAbout 30 pM). The difference in affinity is primarily related to the dissociation rate or the result (resultant) half-life of the complex. For 208F2, the half-life of the complex was 2 to 3 hours for the chimeric and humanized (VH3/VL3) forms. For the other four chimeric antibodies, the average half-life was 7.0 to 9.4 h.

These very slow dissociation kinetics are clearly associated with the bivalent structure of the antibody, which is capable of binding simultaneously to two adjacent h-IGF-1R molecules via their two Fab arms. In this case, the level of trapped IGF-1R molecule may have an effect on the off-rate. The affinities defined in this study correspond to the functional affinity (or avidity) of the antibody at a level of captured h-IGF-1R of about 600 RU. The 3-fold difference in KD observed between the data shown above (table 10) and the values presented in example 13 correlates with a change in the level of capture of hIGF-1R (600RU compared to 160RU in example 4).

Example 14: synthesis of the medicaments of the invention

The following abbreviations are used in the examples below:

aq. aqueous solution

ee enantiomeric excess

equiv equivalent of

ESI electrospray ionization

LC/MS liquid chromatography and mass spectrometry combined use

HPLC high performance liquid chromatography

NMR nuclear magnetic resonance

sat, saturated

UV ultraviolet ray

Reference Compound 1

(S) -2- ((S) -2- ((3-aminopropyl) (methyl) amino) -3-methylbutanamide) -N- ((3R,4S,5S) -3-methoxy-1- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -5-methyl-1-oxoheptan-4-yl) -N, 3-dimethylbutanamide bistrifluoroacetic acid

Compound 1A: (4R,5S) -4-methyl-5-phenyl-3-propionyl-1, 3-oxazolidin-2-one

(4R,5S) -4-methyl-5-phenyl-1, 3-oxazolidin-2-one (5.8g,32.7mmol,1.00equiv) was dissolved in tetrahydrofuran (THF,120mL) under an inert atmosphere. The mixture was cooled to-78 deg.C and n-butyllithium (14.4mL) was added dropwise. After stirring for 30 minutes at-78 deg.C, propionyl chloride (5.7mL) was added. Stirring was continued at-78 ℃ for 30 minutes and then at ambient temperature overnight. The reaction mixture was concentrated and then redissolved in 200mL of water. The pH of the solution was adjusted to 7 with a saturated aqueous solution of sodium bicarbonate. The aqueous phase was extracted 3 times with 100mL ethyl acetate (EtOAc). The organic phases were combined, dried over sodium sulfate, filtered and concentrated to obtain 6.8g (89%) of compound 1A as a yellow oil.

Compound 1B: (2S) -2- [ (1R,2R) -1-hydroxy-2-methyl-3- [ (4R,5S) -4-methyl-2-oxo-5-phenyl-1, 3-oxazolidin-3-yl]-3-oxopropyl radical]Pyrrolidine-1-carboxylic acid tert-butyl ester

Compound 1A (17.6g,75.45mmol,1.00equiv) was dissolved in dichloromethane (DCM,286mL) under an inert atmosphere. The solution was cooled with an ice bath. Triethylamine (TEA,12.1mL,1.15equiv) and Bu were added dropwise₂BOTf (78.3mL,1.04equiv) while maintaining the temperature of the reaction mixture below 2 ℃. Stirring was continued at 0 ℃ for 45 minutes, after which the reaction was cooled to-78 ℃. A solution of tert-butyl (2S) -2-formylpyrrolidine-1-carboxylate (8.5g,42.66mmol,0.57equiv) in DCM (42mL) was added dropwise. Stirring was continued for 2 hours at-78 ℃, then for 1 hour at 0 ℃ and finally for 1 hour at ambient temperature. The reaction was neutralized with 72mL of phosphate buffer (pH 7.2-7.4) and 214mL of methanol, and cooled to 0 ℃. A solution of 30% hydrogen peroxide in methanol (257mL) was added dropwise while maintaining the temperature below 10 ℃. At 0 deg.CStirring was continued for 1 hour. The reaction was neutralized with 142mL of water and then concentrated under reduced pressure. The resulting aqueous solution was extracted 3 times with 200mL EtOAc. The organic phases were combined, dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and petroleum ether (EtOAc: PE ═ 1:8) to give 13.16g (40%) of compound 1B as a colourless oil.

Compound 1C: (2R,3R) -3- [ (2S) -1- [ (tert-butoxy) carbonyl]Pyrrolidin-2-yl radical]-3-hydroxy-2-methylpropionic acid

Compound 1B (13.16g,30.43mmol,1.00equiv) was dissolved in THF (460mL) in the presence of hydrogen peroxide (30% in water, 15.7mL) and then cooled with an ice bath. An aqueous solution of lithium hydroxide (0.4mol/L,152.1mL) was added dropwise while maintaining the reaction temperature below 4 ℃. The reaction mixture was stirred at 0 ℃ for 2.5 hours. Adding Na dropwise₂SO₃(1mol/L,167.3mL) while maintaining the temperature at 0 ℃. The reaction mixture was stirred at ambient temperature for 14 hours, then neutralized with 150mL of cold saturated sodium bicarbonate solution and washed 3 times with 50mL of DCM. With 1M KHSO₄Adjusting the pH of the aqueous solution to 2-3. The aqueous solution was extracted 3 times with 100mL EtOAc. The organic phases were combined, washed once with saturated NaCl solution, dried over sodium sulfate, filtered and concentrated to obtain 7.31g (88%) of compound 1C as colorless oil.

Compound 1D: (2R,3R) -3- [ (2S) -1- [ (tert-butoxy) carbonyl]Pyrrolidin-2-yl radical]-3-methoxy-2-methylpropanoic acid

Compound 1C (7.31g,26.74mmol,1.00equiv) was dissolved in THF (135mL) in the presence of iodomethane (25.3mL) under an inert atmosphere. The reaction medium was cooled with an ice bath, after which NaH (60% in oil, 4.28g) was added in portions.The reaction was left at 0 ℃ for 3 days with stirring, then neutralized with 100mL of a saturated aqueous solution of sodium hydrogencarbonate and washed 3 times with 50mL of diethyl ether. With 1M KHSO₄The aqueous solution of (a) adjusts the pH of the aqueous solution to 3. The aqueous solution was extracted 3 times with 100mL EtOAc. The organic phases were combined and washed with 100mL Na₂S₂O₃(5% in water) and once with a saturated solution of NaCl, then dried over sodium sulfate, filtered and concentrated to obtain 5.5g (72%) of compound 1D as a colorless oil.

Compound 1E: N-methoxy-N-methyl-2-phenylacetamide

2-Phenylacetic acid (16.2g,118.99mmol,1.00equiv) was dissolved in dimethylformamide (DMF,130mL) and then cooled to-10 ℃. Diethyl cyanophosphate (DEPC,19.2mL), methoxy (methyl) amine hydrochloride (12.92g,133.20mmol,1.12equiv) and triethylamine (33.6mL) were added. The reaction mixture was stirred at-10 ℃ for 30 minutes and then at ambient temperature for 2.5 hours. Then, it was extracted twice with 1l EtOAc. The organic phases were combined and washed with 500mL NaHCO₃(sat.) washed twice, once with 400mL of water, then dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:100 to 1:3) to obtain 20.2g (95%) of compound 1E as a yellow oil.

Compound 1F: 2-phenyl-1- (1, 3-thiazol-2-yl) ethan-1-one

Tetramethylethylenediamine (TMEDA,27.2mL) was dissolved in THF (300mL) under an inert atmosphere and then cooled to-78 deg.C before dropwise addition of n-BuLi (67.6mL, 2.5M). 2-bromo-1, 3-thiazole (15.2mL) was added dropwise and stirring was continued at-78 deg.C for 30 min. Compound 1E (25) dissolved in THF (100mL) was added dropwiseg,139.50mmol,1.00 equiv). Stirring was continued at-78 ℃ for 30 minutes and then at-10 ℃ for 2 hours. Using 500mL KHSO₄(sat.) the reaction was neutralized and then extracted 3 times with 1 liter of EtOAc. The organic phases were combined, washed twice with 400mL of water, twice with 700mL of NaCl (sat.), then dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:100 to 1:10) to obtain 25g (88%) of compound 1F as a yellow oil.

Compound 1G: (1R) -2-phenyl-1- (1, 3-thiazol-2-yl) ethan-1-ol

Under an inert atmosphere, a solution of compound 1F (15g,73.8mmol,1.00equiv.) in diethyl ether (300mL) was added dropwise to (+) -B-diisopinocampheylchloroborane ((+) -Ipc₂BCl,110.8 mL). The reaction mixture was stirred at 0 ℃ for 24 hours, then with 300mL NaOH (10% in water) and H₂O₂(30% in water) and finally extracted three times with 500ml of OAc. The organic phases were combined and taken up in 300mL of K₂CO₃(sat.) was washed twice, once with 500mL NaCl (sat.), then dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:20 to 1:2) to obtain 6.3G (42%) of compound 1G as a white solid.

Compound 1H: 2- [ (1S) -1-azido-2-phenylethyl]-1, 3-thiazoles

Compound 1G (6G,29.23mmol,1.00equiv.) was dissolved in THF (150mL) in the presence of triphenylphosphine (13G,49.56mmol,1.70equiv.) under an inert atmosphere, then cooled to 0 ℃. Diethyl azodicarboxylate (DEAD,7.6mL) was added dropwise, followed by diphenyl azidophosphate (DPPA,11mL), then the cooling bath was removed and the solution was allowed to stand at ambient temperature with stirring for 48 hours. The medium was concentrated under reduced pressure. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:100 to 1:30) to obtain 8g of partially purified compound 1H as a yellow oil. Compound 1H was used as such in the following procedure.

Compound 1I: n- [ (1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethyl]Carbamic acid tert-butyl ester

Compound 1H (6.5g,28.2mmol,1.00equiv) was dissolved in THF (100mL) in the presence of triphenylphosphine (6.5g,33.9mmol,1.20equiv) under an inert atmosphere and heated to 50 ℃ for 2 hours. Aqueous ammonia (70mL) was then added and heating continued for 3 hours. The reaction was cooled, neutralized with 500mL of water, and then extracted 3 times with 500mL of EtOAc. The organic phases were combined and extracted twice with 500mL of 1N HCl. The aqueous phases were combined, adjusted to pH 8-9 by addition of sodium hydroxide solution (10% in water), and then extracted 3 times with 500mL of DCM. The organic phases were combined, dried over sodium sulfate, filtered and concentrated to obtain 4.8g (83%) of (1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethan-1-amine as a yellow oil. This compound was then protected with a Boc group ((t-butoxy) carbonyl) to enable its purification. It was dissolved in 1, 4-dioxane (40mL) under an inert atmosphere and then cooled to 0 ℃. Dropwise adding (Boc) diluted in 20mL of 1, 4-dioxane₂O (10.26g,47.01mmol,2.00 equiv). The cold bath was removed, the solution was left overnight at ambient temperature with stirring, then neutralized with 300mL of water and extracted twice with 500mL of EtOAc. The organic phases were combined, dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:100 to 1:20, ee 93%). It was then recrystallized from a hexane/acetone mixture (. about.5-10/1, 1g/10mL) to obtain 6g (84%) of Compound 1I (ee)>99％)。

Compound 1J: (2S) -2- [ (1R,2R) -1-methoxy-2-methyl-2- [ [ (1S) -2-phenyl-1- (1, 3)-Thiazol-2-yl) ethyl]Carbamoyl radical]Ethyl radical]Pyrrolidine-1-carboxylic acid tert-butyl ester

Compound 1I (3g,9.86mmol,1.00equiv) was dissolved in 10mL DCM under an inert atmosphere. Trifluoroacetic acid (TFA,10mL) was added, and the solution was left at ambient temperature overnight with stirring, and then concentrated under reduced pressure to obtain 2.0g (64%) of (1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethan-1-amine trifluoroacetate as a yellow oil. The intermediate was redissolved in 20mL DCM, then compound 1D (1.8g,6.26mmol,1.05equiv), DEPC (1.1g,6.75mmol,1.13equiv) and diisopropylethylamine (DIEA,1.64g,12.71mmol,2.13equiv) were added. The reaction mixture was left at ambient temperature overnight with stirring and then concentrated under reduced pressure. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:100 to 1:3) to obtain 2.3g (81%) of compound 1J as a light yellow solid.

Compound 1K: (2R,3R) -3-methoxy-2-methyl-N- [ (1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethyl]-3- [ (2S) -pyrrolidin-2-yl]Propionamide trifluoroacetate salt

Compound 1J (2.25g,4.75mmol,1.00equiv) was dissolved in 10mL DCM under an inert atmosphere. TFA (10mL) was added and the solution was left overnight at ambient temperature with stirring and then concentrated under reduced pressure to yield 2.18g (94%) of compound 1K as a yellow oil.

Compound 1L: (2S,3S) -2- (benzylamino) -3-methylpentanoic acid

(2S,3S) -2-amino-3-methylpentanoic acid (98.4g,750mmol,1.00equiv) is added portionwise to a 2N sodium hydroxide solution (375mL) at ambient temperature. Benzaldehyde (79.7g,751.02mmol,1.00equiv) was added rapidly and the resulting solution was stirred for 30 minutes. Sodium borohydride (10.9g,288.17mmol,0.38equiv) was added in small portions while maintaining the temperature at 5 to 15 ℃. Stirring was continued for 4 hours at ambient temperature. The reaction mixture was diluted with 200mL of water and then washed twice with 200mL of EtOAc. The pH of the aqueous solution was adjusted to 7 with 2N hydrochloric acid solution. The precipitate formed was collected by filtration to give 149.2g (90%) of compound 1L as a white solid.

Compound 1M: (2S,3S) -2- [ benzyl (methyl) amino]-3-methylpentanoic acid

Compound 1L (25g112.97mmol,1.00equiv) was dissolved in formic acid (31.2g) in the presence of formaldehyde (36.5% 22.3g in water) under an inert atmosphere. The solution was stirred at 90 ℃ for 3 hours and then concentrated under reduced pressure. The residue was triturated in 250mL acetone and then concentrated. The trituration/evaporation procedure was repeated twice with 500mL of acetone to obtain 21.6g (81%) of Compound 1M as a white solid.

Compound 1N: (2S,3S) -2- [ benzyl (methyl) amino]-3-methylpentan-1-ol

LiAlH is added at 0 ℃ in an inert atmosphere₄(0.36g) was suspended in 10mL of THF. Compound 1M (1.5g,6.37mmol,1.00equiv) was added in small portions while maintaining the temperature at 0 to 10 ℃. The reaction mixture was stirred at 65 ℃ for 2 hours and then cooled again to 0 ℃ before the reaction mixture was neutralized by the addition of 360. mu.L of water, 1mL of 15% sodium hydroxide, and 360. mu.L of water in that order. The precipitated aluminium salt was removed by filtration. The filtrate was dried over sodium sulfate, filtered and concentrated. The residue is applied to a silica gel columnA mixture of EtOAc and PE (1:50) was purified to obtain 820mg (58%) of compound 1N as a pale yellow oil.

Compound 1O: (2S,3S) -2- [ benzyl (methyl) amino]-3-methylpentanal

Oxalyl chloride (0.4mL) was dissolved in DCM (15mL) under an inert atmosphere. The solution was cooled to-70 ℃ and a solution of dimethyl sulfoxide (DMSO (0.5mL)) in DCM (10mL) was added dropwise over 15 minutes. The reaction mixture was stirred for 30 minutes, after which a solution of compound 1N (820mg,3.70mmol,1.00equiv) in DCM (10mL) was added dropwise over 15 minutes. The reaction mixture was stirred at low temperature for a further 30 minutes, then triethylamine (2.5mL) was added slowly. The reaction mixture was stirred at-50 ℃ for 1 hour, then the cold bath was removed and the reaction was neutralized with 25mL of water while the temperature was returned to normal. The solution was washed once with 30mL of saturated aqueous NaCl solution, then dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:200) to obtain 0.42g (52%) of compound 1O as a yellow oil.

Compound 1P: (2S,3S) -N-benzyl-1, 1-dimethoxy-N, 3-dimethylpent-2-amine

Compound 1O (4.7g,21.43mmol,1.00equiv) was dissolved in 20mL of methanol at 0 ℃. Concentrated sulfuric acid (4.3mL) was added dropwise and stirring was continued at 0 ℃ for 30 minutes. Trimethyl orthoformate (21.4mL) was added, the cooling bath removed, and the reaction medium was left at ambient temperature with stirring for 3 hours. The reaction medium was diluted with 200mL EtOAc and successively with 100mL 10% Na₂CO₃And 200mL of saturated NaCl, followed by drying over sodium sulfate, filtration and concentration under reduced pressure, to obtain 3.4g (60%) of compound 1P as a pale yellow oil.

Compound 1Q: [ [1- (tert-butoxy) vinyl group]Oxy radical](tert-butyl) dimethylsilane

Diisopropylamine (20g,186.71 mmol,1.08equiv) was dissolved in 170mL THF under an inert atmosphere and cooled to-78 deg.C. nBuLi (2.4M,78.8mL) was added dropwise, the solution stirred at low temperature for 30min (to obtain LDA-lithium diisopropylamide), followed by the addition of tert-butyl acetate (20g,172.18mmol,1.00 equiv). The reaction mixture was stirred at-78 deg.C for 20 minutes, then hexamethylphosphoramide (HMPA,25.8mL), and a solution of tert-butyldimethylsilyl chloride (TBDMSCl,28g,185.80mmol,1.08equiv) in 35mL THF were added. Stirring was continued at low temperature for another 20 minutes, after which the cold bath was removed. The solution was concentrated under reduced pressure. The residue was redissolved in 100mL of water and extracted 3 times with 100mL of PE. The organic phases are combined, washed once with 500mL of saturated aqueous NaCl solution, dried over sodium sulfate, filtered and concentrated. The residue was purified by distillation to obtain 16.6g (83%) of compound 1Q as colorless oil.

Compound 1R: (3R,4S,5S) -4- [ benzyl (methyl) amino]-3-methoxy-5-methylheptanoic acid tert-butyl ester

In an inert atmosphere, compound 1P (2.0g,7.54mmol,1.00equiv) and compound 1Q (2.6g,11.28mmol,1.50equiv) were dissolved in 33mL DCM. The solution was cooled to 0 ℃. DMF (1.2g) and BF were added dropwise₃·Et₂A solution of O (2.1g) in 7.5mL DCM. Stirring was continued for 24 hours at 0 ℃. The reaction medium is washed once with 30mL of sodium carbonate (10%) and twice with 50mL of saturated aqueous solution of NaCl, then dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:100) to obtain 1.82g (91%) of the compound as a yellow oil1R。

Compound 1S: (3R,4S,5S) -3-methoxy-5-methyl-4- (methylamino) heptanoate hydrochloride

Compound 1R (2.4g,6.87mmol,1.00equiv) was dissolved in 35mL of ethanol in the presence of Pd/C (0.12g) and concentrated hydrochloric acid (0.63mL) under an inert atmosphere. The nitrogen atmosphere was replaced by a hydrogen atmosphere and the reaction medium was left for 18 hours at ambient temperature with stirring. The reaction medium is filtered and concentrated under reduced pressure. The residue was triturated in 50mL of hexane, the supernatant was removed, and then the supernatant was dried under reduced pressure to obtain 1.66g (82%) of compound 1S as a white solid.

Compound 1T: (3R,4S,5S) -4- [ (2S) -2- [ [ (benzyloxy) carbonyl]Amino group]-N, 3-dimethylbutyrylamino group]-3-methoxy-5-methylheptanoic acid tert-butyl ester

(2S) -2- [ [ (benzyloxy) carbonyl ] amino ] -3-methylbutanoic acid (15g,0.40mmol,1.00equiv) was dissolved in 300mL DCM in the presence of DIEA (38.3mL) and tripyrrolidinylphosphonium bromide hexafluorophosphate (PyBrOP,32.3 g). The solution was stirred at ambient temperature for 30 minutes, then compound 1S (15.99g,0.42mmol,1.07equiv) was added. The reaction medium is stirred for 2 hours and then concentrated. The residue was purified by reverse phase (C18) with a mixture of Acetonitrile (ACN) and water (30: 70 to 100:0 over 40 minutes) to obtain 17g (58%) of compound 1T as colorless oil.

Compound 1U: (3R,4S,5S) -4- [ (2S) -2-amino-N, 3-dimethylbutanamido]-3-methoxy-5-methylheptanoic acid tert-butyl ester

Compound 1T (76mg,0.15mmol,1.00equiv) was dissolved in 10mL of ethanol in the presence of Pd/C (0.05g) under an inert atmosphere. The nitrogen atmosphere was replaced with a hydrogen atmosphere and the reaction was stirred at ambient temperature for 2 hours. The reaction medium is filtered and concentrated under reduced pressure to yield 64mg of compound 1U as a colorless oil.

Compound 1V: (3R,4S,5S) -4- [ (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl]Amino group]-N, 3-dimethylbutyrylamino group]-3-methoxy-5-methylheptanoic acid ester

Compound 1U (18.19g,50.74mmol,1.00equiv) was dissolved in 400mL of a1, 4-dioxane/water mixture (1:1) in the presence of sodium bicarbonate (12.78g,152mmol,3.00equiv) and 9H-fluoren-9-ylmethyl chloroformate (Fmoc-Cl,19.69g,76mmol,1.50equiv), then stirred at ambient temperature for 2 hours. The reaction medium is then diluted with 500mL of water and extracted 3 times with 200mL of EtOAc. The organic phases were combined, washed once with 200mL of saturated aqueous NaCl solution, dried over sodium sulfate, filtered and concentrated to obtain 40g of partially purified compound 1V as a pale yellow oil.

Compound 1W: (3R,4S,5S) -4- [ (2S) -2- [ [ (9H-fluoren-9-ylmethoxy) carbonyl]Amino group]-N, 3-dimethylbutyrylamino group]-3-methoxy-5-methylheptanoic acid

In a neutral atmosphere, compound 1V (40g,68.88mmol,1.00equiv) was dissolved in 600mL DCM. TFA (300mL) was added. The solution was stirred at ambient temperature for 2 hours and then concentrated under reduced pressure. The residue was purified on a silica gel column with a mixture of methanol and DCM (1:10) to obtain 23.6g (65%) of compound 1W as a colorless oil.

Compound 1X: 9H-fluoren-9-ylmethyl N- [ (1S) -1- [ [ (3R,4S,5S) -3-methoxy-1- [ (2S) -2- [ (1R,2R) -1-methoxy-2-methyl-2- [ [ (1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethyl ] ester]Carbamoyl radical]Ethyl radical]Pyrrolidin-1-yl radical]-5-methyl-1-oxoheptan-4-yl](methyl) carbamoyl group]-2-methylpropyl]Carbamates, their preparation and their use

Compound 1W (2.53g,4.82mmol,1.08equiv) was dissolved in 20mL DCM in the presence of compound 1K (2.18g,4.47mmol,1.00equiv), DEPC (875mg,5.37mmol,1.20equiv) and DIEA (1.25g,9.67mmol,2.16 equiv). The reaction mixture was left at ambient temperature overnight with stirring and then successively saturated with 50mL KHSO₄And 100mL of water, dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of methanol and DCM (1:200 to 1:40) to obtain 2.8g (71%) of compound 1X as a light yellow solid.

Compound 1Y: (2S) -2-amino-N- [ (3R,5S) -3-methoxy-1- [ (2S) -2- [ (1R,2R) -1-methoxy-2-methyl-2- [ [ (1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethyl ] ethyl]Carbamoyl radical]Ethyl radical]Pyrrolidin-1-yl radical]-5-methyl-1-oxoheptan-4-yl]-N, 3-dimethylbutanamide

Compound 1X (2.8g,3.18mmol,1.00equiv) was dissolved in acetonitrile (ACN,12mL) in the presence of piperidine (3mL) and left at ambient temperature with stirring for 18 hours. The reaction was neutralized with 50mL of water and then extracted twice with 100mL of lcm. The organic phases were combined, dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of methanol and DCM (1:100 to 1:40) to obtain 1.2g (57%) of compound 1Y as a yellow solid.

Compound 1ZA: (2S) -2- [ [ (tert-butoxy)) Carbonyl radical](methyl) amino group]-3-methylbutyric acid

(2S) -2- [ [ (tert-butoxy) carbonyl ] amino ] -3-methylbutanoic acid (63g,289.97mmol,1.00equiv) was dissolved in THF (1000mL) in the presence of iodomethane (181mL) under an inert atmosphere. The solution was cooled to 0 ℃ and then sodium hydride (116g,4.83mol,16.67equiv) was added in small portions. The reaction mixture was stirred at 0 ℃ for 1.5 hours, then the cold bath was removed and stirring was continued for 18 hours. The reaction was neutralized with 200mL of water and then concentrated under reduced pressure. The residual aqueous phase was diluted with 4 l of water, washed once with 200mL of EtOAc, and the pH adjusted to 3 to 4 with 1N hydrochloric acid solution. The resulting mixture was extracted 3 times with 1.2L EtOAc. The organic phases are combined, dried over sodium sulfate, filtered and concentrated to obtain 60g (89%) of compound 1ZA as a yellow oil.

Compound 1ZB: (2S) -2- [ [ (tert-butoxy) carbonyl](methyl) amino group]-3-methylbutyric acid benzyl ester

In Li₂CO₃Compound 1ZA (47g,203.21mmol,1.00equiv) was dissolved in DMF (600mL) in the presence of (15.8g,213.83mmol,1.05 equiv). The solution was cooled to 0 ℃ and benzyl bromide (BnBr57.9g,338.53mmol,1.67equiv) was added dropwise. The reaction mixture was left overnight with stirring, then neutralized with 400mL of water and filtered. The resulting solution was extracted twice with 500mL EtOAc. The organic phases were combined, dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:100 to 1:20) to obtain 22.5g (34%) of compound 1ZB as a yellow oil.

Compound 1ZC: (2S) -3-methyl-2- (methylamino) butanoic acid benzyl ester hydrochloride

Compound 1ZB (22.5g,70.00mmol,1.00equiv) was dissolved in 150mL DCM. Gaseous hydrochloric acid was bubbled. The reaction was stirred at ambient temperature for 1 hour, then concentrated under reduced pressure to obtain 17g (94%) of compound 1ZC as a yellow solid.

Compound 1ZD: n- (3, 3-diethoxypropyl) carbamic acid tert-butyl ester

3, 3-Diethoxypentan-1-amine (6g,40.76mmol,1.00equiv) was dissolved in 1, 4-dioxane (30mL) in the presence of TEA (4.45g,43.98mmol,1.08equiv) and then cooled to 0 ℃. Dropwise added (Boc) diluted in 20mL1, 4-dioxane₂O (9.6g,43.99mmol,1.08 equiv). The solution was stirred at 0 ℃ for 2 hours, then at ambient temperature overnight, then neutralized with 10mL of water. The pH was adjusted to 5 with HCl (1%). The solution was extracted 3 times with 50mL EtOAc. The organic phases are combined, dried over sodium sulphate, filtered and concentrated to yield 8.21g (81%) of compound 1ZD as a pale yellow oil.

Compound 1ZE: n- (3-oxopropyl) carbamic acid tert-butyl ester

Compound 1ZD (8.20g,33.15mmol,1.00equiv) was dissolved in 18.75mL of acetic acid and allowed to stand overnight at ambient temperature with stirring. The reaction medium was then extracted 3 times with 30mL EtOAc. The organic phases were combined, washed 3 times with 30mL of saturated NaCl solution, dried over sodium sulfate, filtered and concentrated to obtain 5g (87%) of compound 1ZE as a dark red oil.

Compound 1ZF: (2S) -2- [ (3- [ [ (tert-butoxy) carbonyl ] carbonyl]Amino group]Propyl) (methyl) amino]-3-methylbutyric acid

Compound 1ZE (2.4g,13.86mmol,1.00equiv) was dissolved in 50mL THF in the presence of compound 1ZC (3.56g,13.81mmol,1.00equiv) and DIEA (9.16mL,4.00 equiv). The reaction mixture was stirred at ambient temperature for 30 minutes, then sodium triacetoxyborohydride (5.87g,27.70mmol,2.00equiv) was added. Stirring was continued overnight, then the reaction was neutralized with 100mL of water and extracted 3 times with 50mL of EtOAc. The organic phases were combined, dried over sodium sulfate, filtered and concentrated. The residue was partially purified on a silica gel column with a mixture of EtOAc and PE (1: 4). The crude product obtained is redissolved in 20mL of methanol in the presence of Pd/C (1.2g) and hydrogenated at standard temperature and pressure for 20 minutes. The reaction medium was filtered and concentrated under reduced pressure to obtain 200mg (5%) of compound 1ZF as a white solid.

Compound 1ZG: n- (3- [ [ (1S) -1- [ [ (1S) -1- [ [ (3R,4S,5S) -3-methoxy-1- [ (2S) -2- [ (1R,2R) -1-methoxy-2-methyl-2- [ [ (1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethyl ] ester]Carbamoyl radical]Ethyl radical]Pyrrolidin-1-yl radical]-5-methyl-1-oxoheptan-4-yl](methyl) carbamoyl group]-2-methylpropyl]Carbamoyl radical]-2-methylpropyl](methyl) amino group]Propyl) carbamic acid tert-butyl ester

Compound 1Y (50mg,0.08mmol,1.00equiv) was dissolved in 2mL DMF in the presence of compound 1ZF (26.2mg,0.09mmol,1.20equiv), DIEA (37.7mL) and O- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate (HATU,43.3mg,0.11mmol,1.50 equiv). The reaction was left overnight with stirring at ambient temperature, then diluted with 10mL of water and extracted 3 times with 5mL of EtOAc. The organic phases were combined, dried over sodium sulfate, filtered and concentrated to obtain 100mg of compound 1ZG as a partially purified colorless oil.

Compound 1ZG (90mg,0.10mmol,1.00equiv) was dissolved in 2mL DCM under a neutral atmosphere and the solution was cooled with an ice bath. TFA (1mL) was added, the reaction stirred at ambient temperature for 2 hours, then concentrated under reduced pressure. The residue was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 18% to 31% ACN over 7 min, then from 31% to 100% ACN over 2 min; Waters 2489 UV detector at 254nm and 220 nm). Compound 1 was obtained as a white solid in 25% yield (23 mg).

LC/MS/UV (Atlantis T3 column, 3 μm, 4.6X 100 mM; 35 ℃; 1mL/min, 30% to 60% ACN in water (20 mM ammonium acetate over 6 min); ESI (C)₄₄H₇₃N₇O₆S, accurate mass 827.53) m/z: 829 (MH)⁺),5.84min(93.7％,254nm)。

¹H NMR(300MHz,CD₃OD, ppm) delta (rotamer present) 7.85-7.80(m, 1H); 7.69-7.66(m,1H),7.40-7.10(m,5H),5.80-5.63(m,1H),4.80-4.65(m,2H),4.22-4.00(m,1H),3.89-0.74(m, 58H).

Reference Compound 2

(S) -2- ((S) -2- (((2-aminopyridin-4-yl) methyl) (methyl) amino) -3-methylbutanamido) -N- ((3R,4S,5S) -1- ((S) -2- ((1R,2R) -3- (((1S,2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -3-methoxy-5-methyl-1-oxoheptan-4-yl) -N, 3-dimethylbutanamide trifluoroacetate.

Compound 2A: (S) -2- ((1R,2R) -3- (((1S,2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidine-1-carboxylic acid tert-butyl ester

In an inert atmosphere, compound 1D (2.5g,8.70mmol,1.00equiv) and (1S,2R) -2-amino-1-phenylpropan-1-ol (1.315g,8.70mmol,1.00equiv) were dissolved in DMF (35 mL). The solution was cooled to 0 deg.C, then DEPC (1.39mL) and TEA (1.82mL) were added dropwise. The reaction mixture was stirred at 0 ℃ for 2 hours and then at ambient temperature for 4 hours. The reaction mixture was diluted with 200mL of water and then extracted three times with 50mL of EtOAc. The organic phases were combined and diluted with 50mL KHSO₄(1mol/L) was washed once with 50mL NaHCO₃(sat.) was washed once with 50mL NaCl (sat.), then dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain 3.6g (98%) of Compound 2A as a yellow solid.

Compound 2B: (2R,3R) -N- ((1S,2R) -1-hydroxy-1-phenylpropan-2-yl) -3-methoxy-2-methyl-3- ((S) -pyrrolidin-2-yl) propanamide 2,2, 2-trifluoroacetate

Compound 2A (2.7g,6.42mmol,1.00equiv) was dissolved in DCM (40mL) under an inert atmosphere and then cooled to 0 ℃. TFA (25mL) was added and the solution was stirred at 0 ℃ for 2 h. The reaction mixture was concentrated under reduced pressure to obtain 4.4g of compound 2B as a yellow oil.

Compound 2C: (9H-fluoren-9-yl) methyl ((S) -1- (((3R,4S,5S) -1- ((S) -2- ((1R,2R) -3- (((1S,2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -3-methoxy-5-methyl-1-oxoheptan-4-yl) (methyl) amino) -3-methyl-1-oxobutan-2-yl) carbamate

In an inert atmosphere, compound 2B (4.4g,10.13mmol,1.00equiv) and 1W (5.31g,10.12mmol,1.00equiv) were dissolved in DCM (45 mL). The solution was cooled to 0 ℃ and then DEPC (1.62mL) and DIEA (8.4mL) were added dropwise. The reaction mixture was stirred at 0 ℃ for 2 hours and then at ambient temperature overnight. The reaction mixture was diluted with 100mL of water and extracted three times with 50mL of DCM. The organic phases were combined and diluted with 50mL KHSO₄(1mol/L) was washed once with 50mL NaHCO₃(sat.) was washed once, once with 50mL NaCl (sat.), then dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain 3.3g (39%) of Compound 2C as a yellow solid.

Compound 2D: (S) -2-amino-N- ((3R,4S,5S) -1- ((S) -2- ((1R,2R) -3- (((1S,2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -3-methoxy-5-methyl-1-oxoheptan-4-yl) -N, 3-dimethylbutanamide

Compound 2C (300mg,0.36mmol,1.00eq.) was dissolved in ACN (2mL) and piperidine (0.5mL) under an inert atmosphere. The solution was left overnight at ambient temperature with stirring and then evaporated to dryness under reduced pressure. The residue was purified on a silica gel column with a mixture of DCM and MeOH (1:100) to obtain 150mg (68%) of compound 2D as a white solid.

Compound 2E: 2- ((tert-Butoxycarbonyl) amino) isonicotinic acid methyl ester

Methyl 2-aminopyridine-4-carboxylate (2g,13.14mmol,1.00equiv) was dissolved in tert-butanol (20mL) and di-tert-butyl dicarbonate (4.02g,18.42mmol,1.40equiv) was added. The reaction mixture was stirred at 60 ℃ overnight and then purified by addition of 1M NaHCO₃Aqueous solution (50mL) was used to end the reactionAnd (4) stopping. The solid was recovered by filtration, washed with 50mL EtOH, and then dried under vacuum to obtain 2.5g (75%) of compound 2E as a white solid.

Compound 2F: (4- (hydroxymethyl) pyridin-2-yl) carbamic acid tert-butyl ester

Mixing compound 2E (2.5g,9.91mmol,1.00equiv) with CaCl₂(1.65g) was dissolved in EtOH (30 mL). The solution was cooled to 0 ℃ and then NaBH was added gradually₄(1.13g,29.87mmol,3.01 equiv). The solution was left overnight with stirring at ambient temperature, and then water (50mL) was added to quench the reaction. The mixture was extracted three times with 20mL EtOAc. The organic phases were combined, washed twice with 20mL NaCl (sat.), then dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain 2.0g (90%) of compound 2F as a colorless solid.

Compound 2G: (4-Formylpyridin-2-yl) carbamic acid tert-butyl ester

Compound 2F (2.5g,11.15mmol,1.00equiv) was dissolved in DCE (25mL) and 19.4g MnO was added₂(223.14mmol,20.02 equiv). The mixture was left overnight under stirring at 70 ℃ and then the solid was removed by filtration. The filtrate was evaporated to dryness to obtain 1.4G (57%) of compound 2G as a white solid.

Compound 2H: (S) -benzyl 2- (((2- ((tert-butoxycarbonyl) amino) pyridin-4-yl) methyl) (methyl) amino) -3-methylbutyrate

In combination withThe substances 1ZC (2.93g,11.37mmol,1.10equiv), DIEA (5.39g,41.71mmol,4.03equiv) and NaBH (OAc)₃Compound 2G (2.3G,10.35mmol,1.00equiv) was dissolved in 25mL THF in the presence of (4.39G,20.71mmol,2.00 equiv). The reaction mixture was stirred at ambient temperature for 6 hours, then with 60mL NaHCO₃(sat.) was neutralized and extracted 3 times with 20mL of AcOEt. The organic phases were combined, washed twice with 20mL NaCl (sat.), dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:15) to obtain 2.7g (61%) of compound 2H as a white solid.

Compound 2I: (S) -2- (((2- ((tert-butoxycarbonyl) amino) pyridin-4-yl) methyl) (methyl) amino) -3-methylbutanoic acid

Compound 2H (500mg,1.17mmol,1.00equiv) was dissolved in 10mL of LAcOEt and 2mL of methanol in the presence of Pd/C (250mg) and hydrogenated at ambient temperature and atmospheric pressure for 3 hours. The reaction medium is filtered and concentrated under reduced pressure to yield 254mg (64%) of compound 2I in the form of a colourless solid.

Compound 2J: tert-butyl (4- ((3S,6S,9S,10R) -9- ((S) -sec-butyl) -10- (2- ((S) -2- ((1R,2R) -3- (((1S,2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -2-oxoethyl) -3, 6-diisopropyl-2, 8-dimethyl-4, 7-dioxo-11-oxa-2, 5, 8-triazahodecyl) pyridin-2-yl) carbamate

Compound 2J was prepared in a similar manner to compound 1ZG from amine 2D (85.2mg,0.14mmol,1.50equiv), acid 2I (31.7mg,0.09mmol,1.00equiv), HATU (42.9mg,0.11mmol,1.20equiv), and DIEA (36.7mg,0.28mmol,3.02equiv) in DMF (3 mL). After evaporation to dryness, 100mg of crude product are obtained in the form of a white solid.

Compound 2J (100mg,0.11mmol,1.00equiv) was dissolved in 2mL DCM and 1mL TFA. The reaction was stirred at ambient temperature for 1 hour and then concentrated under reduced pressure. The residue (80mg) was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase; 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10min, then from 40% to 100% ACN over 2 min; Waters 2489 UV detector at 254nm and 220 nm). Compound 2 was obtained as a white solid in 6% yield (6.3 mg).

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6X 100 mm; 40 ℃; 1.8mL/min, from 10% to 95% ACN in water (0.05% TFA) over 6 min)); ESI (C)₄₅H₇₃N₇O₇Accurate mass 823.56) m/z: 824.5 (MH)⁺) And 412.9 (M.2H)⁺/2,100％),3.21min(99.2％,210nm)。

¹H NMR(400MHz,CD₃OD, ppm). delta (rotamer present) 7.81-7.79(m, 1H); 7.39-7.29(m, 5H); 6.61-6.59(m, 2H); 4.84-4.52(m, 1H); 4.32-4.02(m, 1H); 3.90-2.98(m, 10H); 2.90-2.78(m, 1H); 2.55-0.81(m, 39H).

Reference Compound 3

((S) -methyl 2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -N, 3-dimethyl-2- ((S) -3-methyl-2- (methyl (pyridin-4-ylmethyl) amino) butyrylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropionate trifluoroacetate

Compound 3A: (S) -2- ((1R,2R) -1-methoxy-3- (((S) -1-methoxy-1-oxo-3-phenylpropan-2-yl) amino) -2-methyl-3-oxopropyl) pyrrolidine-1-carboxylic acid tert-butyl ester

Compound 1D (3g,10.44mmol,1.00equiv) and methyl (S) -2-amino-3-phenylpropionate (2.25g,12.55mmol,1.20equiv) were dissolved in DMF (40mL) under an inert atmosphere. The solution was cooled to 0 ℃ and DEPC (1.67mL,1.05equiv) and TEA (3.64mL,2.50equiv) were added dropwise. The reaction mixture was stirred at 0 ℃ for 2 hours and then at ambient temperature overnight. The reaction mixture was diluted with 100mL of water and then extracted three times with 50mL of EtOAc. The organic phases were combined and 100mL KHSO was used₄(1mol/L) was washed once with 100mL NaHCO₃(sat.) was washed once with 100mL NaCl (sat.), then dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain 4g (85%) of Compound 3A as a colorless oil.

Compound 3B:2, 2, 2-trifluoroacetic acid salt of methyl (S) -2- ((2R,3R) -3-methoxy-2-methyl-3- ((S) -pyrrolidin-2-yl) propionamido) -3-phenylpropionate

Compound 3A (5g,11.15mmol,1.00equiv) was dissolved in DCM (40mL) under an inert atmosphere. TFA (25mL) was added and the solution was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain 8g of compound 3B as a yellow oil.

Compound 3C: (S) -methyl 2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide) -3-phenylpropionate

Compound 3B (8.03g,17.36mmol,1.00equiv) and 1W (9.1g,17.34mmol,1.00equiv) were dissolved in DCM (80 mL). The solution was cooled to 0 ℃ and DEPC (2.8mL) and DIEA (12mL) were added dropwise. The reaction mixture was stirred at 0 ℃ for 2 hours and then at ambient temperature overnight. The reaction mixture was diluted with 200mL of water and extracted three times with 50mL of DCM. The organic phases were combined and diluted with 50mL KHSO₄(1mol/L) was washed once with 50mL NaHCO₃(sat.) was washed once with 50mL NaCl (sat.), then dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain 5g (34%) of Compound 3C as a yellow solid.

Compound 3D: (S) -methyl 2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2-amino-N, 3-dimethylbutyrylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide-o) -3-phenylpropionate

Compound 3C (5.5g,6.43mmol,1.00equiv) was dissolved in a solution of tetrabutylammonium fluoride (TBAF,2.61g,9.98mmol,1.55equiv) in DMF (100mL) under an inert atmosphere. The solution was stirred at ambient temperature for 2 hours, then diluted with 100mL of water and extracted three times with 50mL of EtOAc. The organic phases were combined, then dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain 3.3g (81%) of compound 3D as a yellow solid.

Compound 3E: (S) -3-methyl-2- (methyl (pyridin-4-ylmethyl) amino) butanoic acid benzyl ester

Pyridine-4-carbaldehyde (1g,9.34mmol,1.00equiv) was dissolved in 10mL of 1, 2-Dichloroethane (DCE) in the presence of compound 1ZC (2.9g,11.25mmol,1.21equiv) and titanium (IV) isopropoxide (4.19mL,1.40 equiv). The mixture was stirred at ambient temperature for 30 minutes, then 2.77g NaBH (OAc) was added₃(13.07mmol1.40 equiv). The reaction medium is left overnight with stirring, then neutralized with 100mL of water and the mixture is extracted 3 times with 50mL of AcOEt. The organic phases were combined and evaporated to dryness. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:20) to yield 1.3g (45%) of compound 3E as a colorless oil.

Compound 3F: (S) -3-methyl-2- (methyl (pyridin-4-ylmethyl) amino) butanoic acid

Compound 3E (800mg,2.56mmol,1.00equiv) was dissolved in 30ml acoet in the presence of Pd/C (300mg) and hydrogenated at ambient temperature and atmospheric pressure for 3 hours. The reaction medium is filtered and concentrated under reduced pressure. The residue was purified on a silica gel column with a mixture of DCM and MeOH (100:1 to 5:1) to obtain 100mg (18%) of compound 3F as a white solid.

Compound 3D (50mg,0.08mmol,1.00equiv) and 3F (26.34mg,0.12mmol,1.50equiv) were dissolved in 3mL DCM. The solution was cooled to 0 ℃ and then 0.018mL DEPC and 0.0392mL DIEA were added. The reaction was stirred at 0 ℃ for 2 hours and then at ambient temperature overnight. The reaction medium was concentrated under reduced pressure and the residue (70mg) was purified by preparative HPLC (Pre-HPLC-001SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 minutes, then from 40% to 100% ACN over 2 minutes; Waters 2545 UV detector at 254nm and 220 nm). Compound 3 was obtained as a white solid in 27% yield (20 mg).

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6X 100 mm; 40 ℃; 1.5mL/min, 10% to 95% ACN in water (0.05% TFA) over 8 min); ESI (C)₄₆H₇₂N₆O₈Accurate mass 836.5) m/z:837.5 (MH)⁺) And 419.4 (M.2H)⁺/2(100％)),7.04min(90.0％,210nm)。

¹H NMR(400MHz,CD₃OD, ppm) delta (rotamer present) 8.76-8.74(m, 2H); 8.53-8.48(m,0.4H, NHCO incomplete exchange); 8.29-8.15(m,0.8H, NHCO incomplete exchange); 8.01(s,2H),7.31-7.22(m,5H),4.88-4.68(m, 3H); 4.31-4.07(m, 2H); 3.94-2.90(m, 18H); 2.55-0.86(m, 38H).

Reference Compound 4

Trifluoroacetic acid salt of (S) -2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -N, 3-dimethyl-2- ((S) -3-methyl-2- (methyl (pyridin-4-ylmethyl) amino) butyrylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropionic acid

Compound 3(100mg,0.11mmol,1.00equiv) was dissolved in a mixture of water (5mL), ACN (5mL) and piperidine (2.5 mL). The reaction mixture was left overnight under stirring and then concentrated under reduced pressure. The residue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters2545 UV detector at 254nm and 220nm) to obtain 20mg (20%) of Compound 4 as a white solid.

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6X 100 mm; 40 ℃; 1.5mL/min, 10% to 95% ACN in water (0.05% TFA) over 8 min); ESI (C)₄₅H₇₀N₆O₈Accurate mass 822.5) m/z: 823.5 (MH)⁺) And 412.4 (M.2H)⁺/2,100％),6.84min(89.1％,210nm)。

¹H NMR (400MHz, CD3OD, ppm) delta (rotamer present) 8.79-8.78(m, 2H); 8.09(m, 2H); 7.30-7.21(m, 5H); 4.80-4.80(m,1H),4.36-0.87(m, 58H).

Reference Compound 6

Methyl (S) -2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3-aminopropyl) (methyl) amino) -3-methylbutanoylamino) -N, 3-dimethylbutanoylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionate bistrifluoroacetate

Compound 6A: (2S) -2- [ (2R) -2- [ (R) - [ (2S) -1- [ (3R,4S,5S) -4- [ (2S) -2- [ (2S) -2- [ (3- [ [ (tert-butoxy) carbonyl ] carbonyl]Amino group]Propyl) (methyl) amino]-3-methylbutanamido group]-N, 3-dimethylbutyrylamino group]-3-methoxy-5-methylheptanoyl]Pyrrolidin-2-yl radical](methoxy) methyl]Propionamido group]-3-Phenylpropionic acid methyl ester

Compound 3D (157.5mg,0.25mmol,1.00equiv) was dissolved in 3mL DCM at 0 ℃ in the presence of carboxylic acid 1ZF (78.7mg,0.27mmol,1.10equiv), DEPC (46 μ l) and DIEA (124 μ l) under an inert atmosphere. The reaction mixture was stirred at low temperature for 2 hours, then the cold bath was removed and stirring was continued for 4 hours. Then concentrated under reduced pressure to obtain 200mg of compound 6A as a crude yellow oil. It is used as such in the following steps.

Compound 6A (200mg,0.22mmol,1.00equiv) was dissolved in 2mL DCM at 0 deg.C under an inert atmosphere. TFA (1mL) was added dropwise and the cooling bath was removed. The reaction mixture was stirred at ambient temperature for 1 hour and then concentrated under reduced pressure. The residue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters 2489 UV detector at 254nm and 220nm) to obtain 60mg (26%, yield of 2 steps) of Compound 6 as a white solid.

LC/MS/UV (Zorbax Eclipse Plus C8,3.5 μm, 4.6X 150 mm; 1mL/min,40 ℃ C., 30 to 80% methanol in water (0.1% H) over 18 minutes₃PO₄))；ESI(C₄₃H₇₄N₆O₈Accurate mass 802.56) m/z: 804 (MH)⁺)；11.50min(91.5％,210nm)。

¹H NMR(300MHz,CD₃OD, ppm): delta (rotamer present) 8.52(d,0.3H, NHCO incomplete exchange); 8.25(d,0.5H, NHCO incomplete exchange); 7.30-7.22 (m, 5H); 4.9-4.6 (m, 3H); 4.2-4.0 (m, 1H); 4.0-0.86 (m, 61H).

Reference Compound 7

Bis-trifluoroacetate salt of (S) -2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3-aminopropyl) (methyl) amino) -3-methylbutanoylamino) -N, 3-dimethylbutanoylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid

Compound 6(70mg,0.08mmol,1.00equiv) was dissolved in a mixture of water (5mL), ACN (2.5mL) and piperidine (5 mL). The reaction mixture was left overnight under stirring at ambient temperature and then concentrated under reduced pressure. The residue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; UVWaters 2489 UV detector at 254nm and 220nm) to afford 14.6mg (21%) of Compound 7 as a white solid.

LC/MS/UV (Ascentis Express C18,2.7 μm, 4.6X 100 mm; 1.5mL/min,40 ℃,0 to 80% methanol in water (0.05% TFA) over 8 min); ESI (C)₄₂H₇₂N₆O₈Accurate mass 788.54) m/z: 790 (MH)⁺),5.71min(96.83％,210nm)。

¹H NMR(300MHz,CD₃OD, ppm): delta (rotamer present) 8.42(d,0.3H, NHCO incomplete exchange); 8.15(d,0.2H, NHCO incomplete exchange); 7.31-7.21 (m, 5H); 4.9-4.6 (m, 3H); 4.25-4.0 (m, 1H); 4.0-0.86 (m, 59H).

Compound 11

(S) -N- ((3R,4S,5S) -3-methoxy-1- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -5-methyl-1-oxoheptan-4-yl) -N, 3-dimethyl-2- ((S) -3-methyl-2- (methyl (4- (methylamino) phenethyl) amino) butyramido) butyramide trifluoroacetate

Compound 11A: n- [4- (2-hydroxyethyl) phenyl]Carbamic acid tert-butyl ester

Di-tert-butyl dicarbonate (16.7g,77mmol,1.05eq.) is added to a solution of 2- (4-aminophenyl) ethanol (10g,72.9mmol,1eq.) in THF (200mL) and the reaction is stirred at ambient temperature overnight. The mixture was diluted with EtOAc (200mL), washed with water (200mL), then 1M HCl (100mL), then saturated NaHCO₃The aqueous solution (100mL) was washed, then washed with brine (100 mL). The organic phase was washed with MgSO₄Dried and then evaporated to dryness under reduced pressure. The crude product was triturated with heptane (150mL) twice and dried in vacuo to give compound 11A as a white solid (14.7g, 84%).

Compound 11B: n- [4- (2-oxoethyl) phenyl]Carbamic acid tert-butyl ester

Compound 11A (2.5g,10.5mmol,1.00equiv) was dissolved in 25mL DCM and then cooled to-78 ℃. A solution of dess-martin periodinane (DMP,6.71g,15.8mmol,1.5equiv) in DCM (10mL) was added dropwise. The cooling bath was removed and stirring was continued at ambient temperature for 1 hour. Saturated aqueous solution of sodium bicarbonate (60mL) and Na₂S₂O₃The reaction was neutralized with a 50/50 mixture of saturated aqueous solution. The resulting solution was extracted 3 times with 30mL EtOAc. The organic phases were combined, washed twice with a saturated aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified over silica gel (EtOAc/PE 1/15) to obtain 1.0g (40%) of compound 11B as a pale yellow solid.

Compound 11C: (2S) -2- [ [2- (4- [ [ (tert-butoxy) carbonyl ] carbonyl]Amino group]Phenyl) ethyl](methyl) amino group]-3-methylbutyric acid benzyl ester

Compound 1ZC (3.5g,13.6mmol,1.1equiv) was dissolved in THF (30mL) in the presence of DIEA (6.4g,49.7mmol,4.0equiv), aldehyde 11B (2.9g,12.3mmol,1.0equiv) and sodium triacetoxyborohydride (5.23g,49.7mmol,2.0 equiv). The reaction mixture was left overnight with stirring at ambient temperature and then neutralized with 60mL of a saturated solution of sodium bicarbonate. The resulting solution was extracted 3 times with 30mL EtOAc. The organic phases were combined, washed twice with a saturated aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified over silica gel (EtOAc/PE 1:20) to obtain 3.7g (68%) of compound 11C as a yellow oil.

Compound 11D: (2S) -2- [ [2- (4- [ [ (tert-butoxy) carbonyl ] carbonyl]Amino group]Phenyl) ethyl](methyl) amino group]-3-methylbutyric acid

Compound 11C (2g,4.5mmol,1equiv) was dissolved in 10mL of methanol in the presence of Pd/C (2g) and hydrogenated at standard temperature and pressure for 2 hours. The reaction medium is filtered and concentrated under reduced pressure to obtain 1.2g (75%) of compound 11D in the form of a yellow oil.

Compound 11E: (2S) -2- [ [2- (4- [ [ (tert-butoxy) carbonyl ] carbonyl](methyl) amino group]Phenyl) ethyl](methyl) amino group]-3-methylbutyric acid

Compound 11D (1.2g,3.4mmol,1.00equiv) was dissolved in THF (20mL) under an inert atmosphere. The reaction medium was cooled with an ice bath, and then NaH (60% in oil, 549mg,13.7mmol,4.0equiv) was added in portions, followed by methyl iodide (4.9g,34mmol,10 equiv). The reaction was left overnight with stirring at ambient temperature, then neutralized with water and washed with 100mL EtOAc. The pH of the aqueous solution was adjusted to 6-7 with 1N HCl. The aqueous solution was extracted 3 times with 100mL EtOAc. The organic phases were combined, dried over sodium sulfate, filtered and concentrated to obtain 800mg (64%) of compound 11E as a yellow solid.

Compound 11F: n- [4- (2- [ [ (1S) -1- [ [ (1S) -1- [ [ (3R,4S,5S) -3-methoxy-1- [ (2S) -2- [ (1R,2R) -1-methoxy-2-methyl-2- [ [ (1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethyl ] ethyl]Carbamoyl radical]Ethyl radical]Pyrrolidin-1-yl radical]-5-methyl-1-oxoheptan-4-yl](methyl) carbamoyl group]-2-methylpropyl]Carbamoyl radical]-2-methylpropyl](methyl) amino group]Ethyl) phenyl]-N-methylcarbamic acid tert-butyl ester

Compound 11F was prepared in a similar manner to compound 6A from amine 1Y (150mg,0.22mmol,1.2equiv) and acid 11E (70mg,0.19mmol,1.0 equiv). After purification on silica gel (EtOAc/PE 1:1), 100mg (52%) of the desired product are obtained in the form of a pale yellow solid.

Compound 11 was prepared from intermediate 11F (100mg,0.1mmol) in the same manner as compound 1. The residue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters 2489 UV detector at 254nm and 220 nm). Compound 11 was obtained as a white solid in 39% yield (39.7 mg).

LC/MS/UV (Eclipse Plus C8,3.5 μm, 4.6X 150 mm; 1mL/min,40 ℃,50 to 95% methanol in water (0.05% TFA) over 18 min); ESI (C)₅₀H₇₇N₇O₆S, accurate mass 903.57) m/z: 904.5 (MH)⁺),7.53min(93.68％,254nm)。

¹H NMR(300MHz,CD₃OD, ppm): delta (rotamer present) 8.84(d,0.5H, NHCO incomplete exchange); 8.7-8.5 (m,0.9H, NHCO incomplete exchange); 7.76-7.73 (m, 1H); 7.55-7.4(m, 1H); 7.28-7.22 (m, 7H); 7.08-7.05 (m, 2H); 5.51-5.72 (m, 1H); 4.9-4.80 (m, 2H); 4.3-0.7 (m, 60H).

Compound 12

(S) -methyl 2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -N, 3-dimethyl-2- ((S) -3-methyl-2- (methyl (4- (methylamino) phenethyl) amino) butyrylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropionate trifluoroacetate

Compound 12 was prepared in the same manner as the last stage of the synthesis of Compound 1 from amine 3D (118mg,0.19mmol) and acid 11E (82mg,0.22mmol) in two steps. The final residue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters 2489 UV detector at 254nm and 220 nm). Compound 12 was obtained as a white solid in 7% yield (13.7 mg).

LC/MS/UV (Eclipse Plus C8,3.5 μm, 4.6X 150 mm; 1mL/min,40 ℃,40 to 95% methanol in water (0.05% TFA) over 18 min); ESI (C)₄₉H₇₈N₆O₈Accurate mass 878.59) m/z: 879.7 (MH)⁺),10.07min(90.6％,254nm)。

¹NMR (300MHz, CD3OD, ppm). delta. (rotamer present) 7.40(se, 2H); 7.38-7.22 (m, 7H); 4.95-4.7 (m, 3H); 4.2-4.0 (m, 1H); 3.9-0.86 (m, 62H).

Compound 13

Trifluoroacetic acid salt of (S) -2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -N, 3-dimethyl-2- ((S) -3-methyl-2- (methyl (4- (methylamino) phenethyl) amino) butyrylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropionic acid

Compound 13 was prepared from compound 12(100mg,0.10mmol) in the same manner as compound 7. The residue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters 2489 UV detector at 254nm and 220 nm). Compound 13 was obtained as a white solid in 20% yield (20 mg).

LC/MS/UV (Ascentis Express C18,2.7 μm, 4.6X 100 mm; 1.5mL/min,40 ℃,10 to 95% methanol in water (0.05% TFA) over 8 min); ESI (C)₄₈H₇₆N₆O₈Accurate mass 864.57) m/z: 865.6 (MH)⁺),6.05min(90.9％,210nm)。

¹H NMR (300MHz, CD3OD, ppm): delta (rotamers present) 7.32-7.19 (m, 9H); 4.9-4.65 (m, 3H); 4.2-4.0 (m, 1H); 3.9-0.86 (m, 59H).

Compound 14

(S) -2- ((S) -2- ((3-aminobenzyl) (methyl) amino) -3-methylbutanamide) -N- ((3R,4S,5S) -3-methoxy-1- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -5-methyl-1-oxoheptan-4-yl) -N, 3-dimethylbutyramide trifluoroacetate

Compound 14A: (3- (hydroxymethyl) phenyl) carbamic acid tert-butyl ester

(3-aminophenyl) methanol (3g,24.36mmol,1.00equiv) was dissolved in THF (60mL), followed by the addition of di-tert-butyl dicarbonate (6.38g,29.23mmol,1.20 equiv). The reaction mixture was left overnight with stirring at ambient temperature and then diluted by the addition of 200mL of water. The product was extracted 3 times with 100mL AcOEt, then the organic phases were recombined, dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product as a yellow oil (13.85g compound 14A).

Compound 14B: (3-formylphenyl) carbamic acid tert-butyl ester

Compound 14A (13.8g,61.81mmol,1.00equiv) was dissolved in DCE (400mL) and MnO was added₂(54g,621.14mmol,10.05 equiv). The mixture was left under stirring at ambient temperature for 3 days, then the solids were removed by filtration. The filtrate was evaporated to dryness and the residue was purified on a silica gel column with a mixture of EtOAc and PE (1:30) to obtain 3g (22%) of compound 14B as a white solid.

Compound 14C: (S) -benzyl 2- ((3- ((tert-butoxycarbonyl) amino) benzyl) (methyl) amino) -3-methylbutyrate

In the presence of compound 1ZC (1.16g,4.50mmol,1.00equiv), DIEA (3mL) and NaBH (OAc)₃Compound 14B (1g,4.52mmol,1.00equiv) was dissolved in 20mL THF in the presence of (1.92g,9.06mmol,2.01 equiv). The reaction mixture was left overnight with stirring at ambient temperature, then neutralized with 100mL of water and extracted 3 times with 50mL of AcOEt. The organic phases were combined, dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column with a mixture of EtOAc and PE (1:50) to obtain 1.9g (99%) of compound 14C as a white solid.

Compound 14D: (S) -2- ((3- ((tert-butoxycarbonyl) amino) benzyl) (methyl) amino) -3-methylbutanoic acid

Compound 14C (1g,2.34mmol,1.00equiv) was dissolved in 30mL of LAcOEt and 4mL of methanol in the presence of Pd/C (400mg) and hydrogenated at ambient temperature and atmospheric pressure for 1 hour. The reaction medium was filtered and concentrated under reduced pressure to obtain 680mg (86%) of compound 14D as a white solid.

Compound 14E: (3- ((3S,6S,9S,10R) -9- ((S) -sec-butyl) -3, 6-diisopropyl-10- (2- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -2-oxoethyl) -2,8-dimethyl-4, 7-dioxo-11-oxa-2, 5, 8-triazodecyl) phenyl) carbamic acid tert-butyl ester

Compound 14E was synthesized in the same manner as compound 3 from amine 1Y (100mg,0.15mmol,1.00equiv), acid 14D (102.27mg,0.30mmol,2.00equiv), DEPC (0.053mL) and DIEA (0.046mL) in DCM (3 mL). The crude product (80mg) was purified on a silica gel column with a mixture of EtOAc and PE (1:1) to afford 100mg (67%) of compound 14E as a light yellow solid.

Compound 14 was prepared from intermediate 14E (100mg,0.10mmol,1.00equiv) in the same manner as compound 2. The crude product (80mg) was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters2545 UV detector at 254nm and 220 nm). Compound 14 was obtained as a white solid in 10% yield (10 mg).

LC/MS/UV (Eclipse plus C8 column, 3.5 μm, 4.6X 150 mm; 40 ℃; 1.0mL/min, 40% to 95% MeOH in water (0.05% TFA) over 18 min)); ESI (C)₄₈H₇₃N₇O₆S, accurate mass 875.5) m/z: 876.5 (MH)⁺) And 438.9 (M.2H)⁺/2,100％),11.35min(95.6％,210nm)。

¹H NMR(400MHz,CD₃OD, ppm). delta (rotamer present) 8.92-8.86(m,0.4H, NH incomplete exchange); 8.70-8.54(m,0.6H, NH incomplete exchange); 7.88-7.78(m, 1H); 7.60-7.50(m, 1H); 7.45-6.97(m, 9H); 5.80-5.65(m, 1H); 4.85-4.70(m, 1H); 4.40-0.80(m, 56H).

Compound 15

Methyl (S) -2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3-aminobenzyl) (methyl) amino) -3-methylbutanoylamino) -N, 3-dimethylbutanoylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionate trifluoroacetate

Compound 15A: (S) -methyl 2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3- ((tert-butoxycarbonyl) amino) benzyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropionate

Compound 15A was synthesized in the same manner as compound 3 from amine 3D (200mg,0.32mmol,1.00equiv), acid 14D (212.6mg,0.63mmol,2.00equiv), DEPC (0.1103mL) and DIEA (0.157mL,3.00equiv) in DCM (5 mL). The crude product was purified on a silica gel column with a mixture of EtOAc and PE (1:1) to obtain 200mg (67%) of compound 15A as a yellow solid.

Compound 15: compound 15 was synthesized from intermediate 15A (200mg,0.21mmol,1.00equiv) in the same manner as compound 2. The crude product was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters2545 UV detector at 254nm and 220 nm). Compound 15 was obtained as a white solid in 19% yield (38.6 mg).

LC/MS/UV (Ascentis Express C18 column, 2.7 μm, 4.6X 100 mm; 40 ℃; 1.5mL/min, 10% to 95% MeOH in water (0.05% TFA) over 8 min); ESI (C)₄₇H₇₄N₆O₈Accurate mass 850.5) m/z: 851.5 (MH)⁺) And 426.4 (M.2H)⁺/2,100％),6.61min(91.1％,210nm)。

¹H NMR(400MHz,CD₃OD, ppm) delta (rotamer present) 7.53-7.42(m, 1H); 7.35-7.18(m, 8H); 4.88-4.79(m, 2H); 4.42-4.00(m, 3H); 3.93-2.71(m, 22H); 2.61-0.81(m, 33H).

Compound 20

(S) -2- ((S) -2- ((4-aminobenzyl) (methyl) amino) -3-methylbutanamide) -N- ((3R,4S,5S) -3-methoxy-1- ((S) -2- ((1R,2R) -1-methoxy-2-methoxy-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -5-methyl-1-oxoheptan-4-yl) -N, 3-dimethylbutyramide trifluoroacetate

Compound 20 was prepared from amine 1ZC and the corresponding aldehyde in the same manner as compound 1.

The 4-nitrobenzaldehyde involved in the preparation of compound 20 is commercially available.

The synthesis of compound 20 was completed by reduction of the nitro group. The synthesis proceeds as follows: (2S) -N- [ (3R,4S,5S) -1- [ (2S) -2- [ (1R,2R) -2- [ [ (1S,2R) -1-hydroxy-1-phenylpropan-2-yl ] carbamoyl ] -1-methoxy-2-methylethyl ] pyrrolidin-1-yl ] -3-methoxy-5-methyl-1-oxoheptan-4-yl ] -N, 3-dimethyl-2- [ (2S) -3-methyl-2- [ methyl [ (4-nitrophenyl) methyl ] amino ] butanamide (40mg,0.05mmol,1.0equiv) was dissolved in 15mL of ethanol. Tin (II) chloride dihydrate (317mg,1.4mmol,30equiv) was added and the solution was left at ambient temperature with stirring for 3 days. The reaction was neutralized with 50mL of water and then extracted three times with 50mL of EtOAc. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain compound 20 (purity: 93.2%; amount: 21.6mg) in a crude state.

The compound was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5 μm,19 × 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters 2489 uv detector at 254nm and 220nm) to obtain the corresponding TFA salt as a white solid.

¹H NMR:(400MHz,CD₃OD, ppm) delta (rotamer present) 7.85-7.80 (m, 1H); 7.6-7.5 (m, 1H); 7.4-7.15 (m, 5H); 7.1-7.05 (m, 2H); 6.73-6.70 (m, 2H); 5.8-5.55 (m, 1H); 5.0-4.7 (m, 2H); 4.25-4.05 (m, 1H); 4.0-0.8 (m, 54H). LC/MS/UV ESI (C)₄₈H₇₃N₇O₇S, accurate Mass 875.53) m/z876 (MH)⁺),439[75％,(M.2H⁺)/2]；UV:RT＝4.83min(96.8％,254nm)。¹H NMR(400MHz,CD₃OD, ppm) delta (rotamer present) 7.85-7.80 (m, 1H); 7.6-7.5 (m, 1H); 7.4-7.1 (m, 7H); 6.76-6.72 (m, 2H); 5.8-5.55 (m, 1H); 4.9-4.65 (m, 2H); 4.25-4.05 (m, 1H); 4.0-0.8 (m, 54H).

Compound 29

Trifluoroacetate salt of (S) -2- ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3-aminobenzyl) (methyl) amino) -3-methylbutanoylamino) -N, 3-dimethylbutanoylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoylamino) -3-phenylpropionic acid

Compound 15(100mg,0.10mmol,1.00equiv) was dissolved in a mixture of water (5mL), ACN (5mL) and piperidine (2.5 mL). The reaction mixture was left overnight under stirring at ambient temperature and then concentrated under reduced pressure. The residue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunAire Prep C18 OBD column, 5 μm, 19X 150 mm; elution phase: 0.05% TFA buffered water/ACN; gradient from 20% to 40% ACN over 10 min, then from 40% to 100% ACN over 2 min; Waters2545 UV detector at 254nm and 220nm) to obtain 20mg (20%) of compound 29 as a white solid.

LC/MS/UV (Eclipse Plus C8 column, 3.5 μm, 4.6X 150 mm; 40 ℃; 1.0mL/min, 40% to 95% MeOH in water (0.05% TFA) over 18 min)); ESI (C)₄₆H₇₂N₆O₈Accurate mass 836.54) m/z 837.5 (MH)⁺) And 419.4 (M.2H)⁺/2,100％),10.61min(92.5％,210nm)。

¹H NMR:(400MHz,CD₃OD, ppm) delta (rotamer present) 7.38-7.15(m, 6H); 7.00-6.99(m, 3H); 4.85-4.68(m, 2H); 4.37-3.38(m, 11H); 3.31-2.70(m, 8H); 2.60-0.82(m, 35H).

Compound 61

(S) -2- ((S) -2- ((4-Aminophenethyl) (methyl) amino) -3-methylbutanamido) -N- ((3R,4S,5S) -3-methoxy-1- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -5-methyl-1-oxoheptan-4-yl) -N, 3-dimethylbutanamide

Compound 61A: n- (4-Aminophenylethyl) -N-methyl-L-valine dihydrochloride

Compound 11D (962mg,2.75mmol) was dissolved in 10mL of a commercially available solution of HCl in propan-2-ol (5-6M) and stirred at room temperature for 2 h. TLC analysis indicated complete consumption of the starting material. The solvent was evaporated under reduced pressure and Et₂The resulting yellow solid was triturated O (2X 10 ml). The product was dried in vacuo to give compound 61A as a yellow solid (322mg, 47%).

Compound 61: carboxylic acid 61A (73mg,0.23mmol,1eq.) and amine 1Y (150mg,0.23mmol,1eq.) were dissolved in dry DMF (2 ml). DIEA (158. mu.l, 0) was added90mmol,4eq.) and DECP (51. mu.l, 0.34mmol,1.5eq.) and the reaction was stirred at room temperature for 4 hours. LC-MS analysis indicated complete consumption of the starting material. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography on silica gel (DCM/MeOH) to give compound 61 as a pale yellow solid (83mg, 40%).

¹H NMR:(500MHz,DMSO-d₆Ppm). delta (rotamer present), 8.86(d,0.5H, NHCO); 8.65(d,0.5H, NHCO),8.11-8.05(m,1H, NHCO),7.80(d,0.5H, thiazole), 7.78(d,0.5H, thiazole), 7.65(d,0.5H, thiazole), 7.63(d,0.5H, thiazole), 7.32-7.12 (m,5H),6.83(d, J ═ 8.3Hz,2H),6.45(d, J ═ 8.3Hz,2H), 5.56-5.49 (m,0.5H), 5.42-5.35 (m,0.5H),4.78(s,2H, NH2), 4.74-4.46 (m,2H), 4.01-0.66 (m, 57H).

HPLC (Xbridge Shield C18,3.5 μm, 4.6X 50 mm; 3.5ml/min,40 ℃,0 to 95% MeCN in water (0.1% TFA) over 2.25 min, then 95% MeCN for 0.5 min, Tr 1.31min (96.5%, 220 nm).

m/z(Q-TOF ESI⁺)890.5558(2％,MH⁺,C₄₉H₇₆N₇O₆S requirement 890.5572),445.7834 (100%, (MH)₂)²⁺,C₄₉H₇₇N₇O₆S requires 445.7823).

Compound 62

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4-aminophenylethyl) (methyl) amino) -3-methylbutanoylamino) -N, 3-dimethylbutanoylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester

Compound 62 was prepared in the same manner as compound 61 using carboxylic acid 61A (69mg,0.21mmol,1eq.), amine 3D (135mg,0.21mmol,1eq.), DIEA (75 μ l,0.43mmol,2eq.), and DECP (49 μ l,0.32mmol,1.5 eq.). The crude product was purified by flash chromatography on silica gel (DCM/MeOH) to give compound 62 as a yellow solid (82mg, 45%).

¹H NMR:(500MHz,DMSO-d₆δ (rotamer present), 8.50(d, J ═ 8.3,0.5H, NHCO); 8.27(d, J ═ 8.0,0.5H, NHCO),8.15-8.04(m,1H, NHCO), 7.27-7.13 (m,5H), 6.86-6.79 (m,2H), 6.48-6.42 (m,2H),4.78(s,2H, NH)₂),4.74–4.44(m,3H),4.01–3.72(m,1.5H),3.66(s,1.5H,CO₂Me),3.63(s,1.5H,CO₂Me),3.57-0.65(m,55.5H)。

HPLC (Xbridge Shield C18,3.5 μm, 4.6X 50 mm; 3.5ml/min,40 ℃,0 to 95% MeCN in water (0.1% TFA) over 2.25 min, then 95% MeCN for 0.5 min, Tr 1.29min (95.3%, 220 nm).

m/z(Q-TOF ESI⁺)865.5800(2％,MH⁺,C₄₈H₇₇N₆O₈Requirements 865.5797),433.2937 (100%, (MH)₂)²⁺,C₄₈H₇₈N₆O₈Requirement 433.2935).

Compound 63

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4-aminophenylethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine 2,2, 2-trifluoroacetate

Compound 62(23mg,0.03mmol) was dissolved in a mixture of water (1ml) and acetonitrile (1 ml). Piperidine (0.75ml) was added to the solution, and the mixture was stirred at room temperature for 5 hours. TLC analysis indicated complete consumption of the starting material. The solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC (SunAire Prep column C18 OBD,5 μm, 19X 150 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 20% to 40% MeCN over 10 min, then from 40% to 100% MeCN over 2 min; UV detector Waters2545 at 254nm and 220 nm). Compound 63(14mg, 66%) was obtained as a white solid.

¹H NMR:(500MHz,DMSO-d₆In ppm delta (rotamer present), 12.7(s (br),1H, CO₂H),9.58(m(br),1H)；9.04–8.89(m,1H),8.41(d,0.6H,NHCO),8.15(d,0.4H,NHCO),7.27–7.13(m,5H),7.13–6.99(m(br),2H),6.90–6.64(s(br),2H),4.77–3.40(m,10H),3.34–2.75(m,20H),2.34–1.94(m,4H),1.90–0.7(m,25H)。

HPLC (Xbridge Shield C18,3.5 μm, 4.6X 50 mm; 3.5ml/min,40 ℃,0 to 95% MeCN in water (0.1% TFA) over 2.25 min, then 95% MeCN for 0.5 min, Tr 1.24min (100%, 220 nm).

m/z(Q-TOF ESI⁺)851.5641(6％,MH⁺,C₄₇H₇₅N₆O₈Requirements 851.5641),426.2854 (100%, (MH)₂)²⁺,C₄₇H₇₆N₆O₈Requirement 426.2857).

Example 15: antiproliferative activity of drugs

The method comprises the following steps:

and (5) culturing the cells. A549 (non-small cell lung carcinoma-ATCC CCL-185) and MDA-MB-231 (breast cancer-ATCCHTB-26) cells were cultured in minimal essential Medium Eagle (MEM) containing 5% Fetal Calf Serum (FCS) and Dulbecco's Modified Eagle Medium (DMEM) containing 10% FCS, respectively. MCF7 (ductal carcinoma of the breast-ATCC HTB-22) and SN-12C (renal carcinoma-ATCC) cells were maintained in RPMI1640 medium with 10% FCS (no phenol red for MCF7 cells). All media were supplemented with amphotericin (1.25. mu.g/mL) and penicillin-streptomycin (100U/100. mu.g/mL). 5% CO at 37 ℃ in an incubator under standard conditions₂And culturing the cells at 95% atmospheric humidity.

Antiproliferative activity in 4 tumor cell lines. The ATPLite proliferation assay (Perkin Elmer, Villebon sur Yvette, France) was used at 4The antiproliferative activity of the selected drugs was investigated on a panel of seed cell lines. Cells were seeded on 96-well plates on day 0(10 for a 549)³Cells/well, 2.10 for MCF7, MDA-MB-231, and SN12C³) The concentration of the compound can ensure that the cells are in the logarithmic cell growth phase in the 72h drug treatment phase. After 24h incubation period, all cells were treated with serial dilutions of test compound (11 μ Ι _ of 10X solution in 1% DMSO-6 wells/condition). To avoid cell adhesion to the tip (tip), the tip was changed between two serial dilutions. Then, the cells were placed at 37 ℃ in 5% CO₂In an incubator. On day 4, cell viability was assessed by measuring (dose) ATP released by viable cells. The number of viable cells was analyzed in comparison to the number of solvent-treated cells. EC determination using curve fitting analysis (non-linear regression model with sigmoidal dose response, variable slope coefficient)₅₀Values, the curve fitting analysis was performed using an algorithm provided by GraphPad Software (GraphPad Software inc., CA, usa).

As a result:

various drugs:

various drugs were tested to determine their antiproliferative activity in the MDA-MB-231 cell line according to the methods described above. Measured activity gives the EC₅₀<A value of 0.1. mu.M.

Several of the following examples selected from the above exemplified drugs illustrate their fully significant anti-proliferative properties:

example 12: EC (EC)₅₀＝5.80×10^-10M; example 13: EC (EC)₅₀＝7.95×10^-8M; example 15: EC (EC)₅₀＝1.70×10^-10M; example 27: EC (EC)₅₀＝1.20×10^-10M。

Various cell lines:

according to the method described above, in different cell lines (A549, MDA-MB-231, MCF-7, M-B-231, M-B-D-,SN12C) compound 15. The measured activity gives the EC in all cell lines tested₅₀<A value of 0.1. mu.M.

EC₅₀(M)	A549	MDA-MB-231	MCF-7	SN12C
					Compound 15	1.45x10^-10	1.70x10^-10	7.15x10^-10	2.18x10^-10

Comparative example:

the substitution on the phenyl ring (amino group compared to carboxyl group) was investigated in the following comparative examples, showing the improved antiproliferative activity of the drugs according to the invention comprising an amino substituent.

Example 16: synthesis of drug-linker moieties

Compound E-11

4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl (4- ((3R,4S,7S,10S) -4- ((S) -sec-butyl) -7, 10-diisopropyl-3- (2- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 11-dimethyl-6, 9-dioxo-2-oxa-5, 8, 11-triazatridecan-13-yl) phenyl) (methyl) carbamate 2,2, 2-trifluoroacetate salt

Compound E-11-1: (S) -2-amino-5-ureidopentanoic acid methyl ester hydrochloride

Acetyl chloride (10mL) was added dropwise to MeOH (120mL) with stirring at 0 deg.C. After 20min, L-citrulline (10g, 57mmol, 1.00eq.) was added and the mixture was heated under reflux overnight. The solvent was evaporated under reduced pressure to obtain 15g (116%) of compound E-11-1 as a white solid. The product was used in the next step without further drying.

Compound E-11-2: (S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanoic acid methyl ester

Compound E-11-1(13g,57.6mmol,1.1eq.) was dissolved in DMF (140mL) at 0 ℃ under an inert atmosphere. DIEA (30mL,173mmol,3.0eq.) and hydroxybenzotriazole (HOBt-10.59g,69.1mmol,1.2eq.) and Boc-L-valine hydroxysuccinimide ester (Boc-Val-OSu-18.1) were addedg,57.6mmol,1.0 eq.). The reaction mixture was stirred at ambient temperature overnight, then the solvent was evaporated under reduced pressure. The residue was dissolved in water (100mL) and extracted twice with DCM (150 mL). The organic phases were combined with Na₂SO₄Dried and concentrated under reduced pressure. The residue was purified on silica gel (DCM/MeOH) to obtain 18.8g (84%) of compound E-11-2 as a white solid.

Compound E-11-3: (S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamide) -5-ureidopentanoic acid

Compound E-11-2(18.8g,48.4mmol,1eq.) was dissolved in MeOH (200mL) at 0 ℃. A 1m naoh solution (72mL,72mmol,1.5eq.) was added and the mixture was stirred at room temperature for 2 hours. MeOH was removed under reduced pressure and the residual aqueous solution was acidified using 1 MHCl. The aqueous phase was evaporated to dryness and the residue was purified on silica gel (DCM/MeOH) to obtain 18g (99%) of compound E-11-3 as a white solid.

Compound E-11-4: ((S) -1- (((S) -1- ((4- (hydroxymethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamic acid tert-butyl ester

Compound E-11-3(5g,13.4mmol,1eq.) was dissolved in a mixture of dry DCM (65ml) and dry MeOH (35 ml). (4-aminophenyl) methanol (1.81g,14.7mmol,1.1eq.) and N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ-6.60g,26.7mmol,2eq.) were added and the mixture was stirred overnight in the dark. The solvent was evaporated under reduced pressure and the residue was purified on silica gel (DCM/MeOH) to obtain 5.2g (73%) of compound E-11-4 as an off-white solid.

Compound E-11-5: ((S) -3-methyl-1- (((S) -1- ((4-, (((4-Nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -1-oxobutan-2-yl) carbamic acid tert-butyl ester

Compound E-11-4(1.1g,2.29mmol,1eq.) was dissolved in dry DMF (5mL) at ambient temperature under an inert atmosphere. Bis (4-nitrophenyl) carbonate (1.40g,4.59mmol,2eq.) was added followed by DIEA (600 μ l,3.44mmol,1.5eq.) and the resulting yellow solution was stirred overnight. DMF was evaporated under reduced pressure and the residue was purified on silica gel (DCM/MeOH) to give 1.27g (84%) of compound E-11-5 as an off-white solid.

Compound E-11-6: 4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) benzyl (4- ((3R,4S,7S,10S) -4- ((S) -sec-butyl) -7, 10-diisopropyl-3- (2- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 11-dimethyl-6, 9-dioxo-2-oxa-5, 8, 11-triazatridecan-13-yl) phenyl) (methyl) carbamate 2,2, 2-trifluoroacetate salt

Carbonate E-11-5(114mg,0.177mmol,1.2eq.) and aniline 11F (150mg,0.147mmol,1eq.) were dissolved in dry DMF (4 mL). HOBt (38mg,0.295mmol,2eq.) and DIEA (54 μ L,0.295mmol,2eq.) were added and the mixture was stirred at room temperature over the weekend. DMF was evaporated under reduced pressure and the residue was purified by flash chromatography on silica gel, eluting with DCM. The product was re-purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound E-11-6 as a white solid (89mg, 39%).

Compound E-11：

Compound E-11-6(21mg,0.014mmol,1.0eq.) was dissolved in DCM (0.25mL) and TFA (40 μ L) was added. The solution was stirred at room temperature for 2 hours, after which LC-MS analysis indicated complete consumption of the starting material. The mixture was cooled briefly (liquid nitrogen bath) while DMF (0.5mL) was added, followed by DIEA (100. mu.L) to neutralize the TFA. The cooling bath was then removed and 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (4mg,0.012mmol,1eq.) was added. The mixture was stirred at room temperature for 48 h and the product was purified by preparative HPLC (Waters600E, SunFirep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound E-11 as a white solid (11mg, 54%).

m/z(Q-TOF MS ESI+)1524.8282(2％,MNa⁺,C₇₉H₁₁₅N₁₃NaO₁₄S requirement 1524.8299),751.9283 (100%, (MH)₂)²⁺,C₇₉H₁₁₇N₁₃O₁₄S requires 751.9276).

Compound E-12

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- (((4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) (methyl) amino) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2- Methylpropanoyl) -L-phenylalanine methyl ester 2,2, 2-trifluoroacetate salt

Compound E-12-1: ((S) -3-methyl-1-oxo-1- (((S) -1-oxo-1- ((4- ((((perfluorophenoxy) carbonyl) oxy) methyl) phenyl) amino) -5-ureidopentan-2-yl) amino) butan-2-yl) carbamic acid tert-butyl ester

Compound E-11-4(670mg,1.26mmol,1eq.) was dissolved in dry DMF (6mL) at 0 ℃ under an inert atmosphere. Bis (perfluorophenyl) carbonate (991mg,2.51mmol,2eq.) was added followed by DIEA (329 μ l,1.89mmol,1.5eq.) and the resulting colorless solution was stirred at room temperature for 30 minutes. DMF was evaporated under reduced pressure and the residue was purified on silica gel (DCM/MeOH) to give 836mg (96%) of compound E-12-1 as an off-white solid.

Compound E-12-2: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((((4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) (methyl) amino) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester 2,2, 2-Trifluoroacetate salt

Aniline 12(165mg,0.189mmol,1.0eq.) was dissolved in DMF (5mL) at 0 ℃ under an inert atmosphere. Carbonate E-12-1(194mg,0.282mmol,1.5eq.) HOBt (51mg,0.375mmol,2eq.) and DIEA (66. mu.L, 0.375mmol,2eq.) were added and the mixture was stirred at room temperature for 8 hours. The solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound E12-7 as a white solid (247mg, 77%).

Compound E-12-3: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((((4- ((S) -2- ((S) -2-amino-3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) (methyl) amino) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester bis (2,2, 2-trifluoroacetic acid salt

Compound E-12-2(5.6mg, 4.04. mu. mol,1.0eq.) was dissolved in TFA (100. mu.L). After 5 minutes, 2ml of water was added and the mixture was lyophilized overnight to give compound E-12-3 as an off-white solid (5.6mg, 98%).

Compound E-12：

Compound E-12-3(5.6mg,4 μmol,1.0eq.) was dissolved in acetonitrile (0.5mL) and DIEA (5 μ L,7eq.) was added followed by 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (2.5mg,8 μmol,2 eq.). The mixture was stirred at room temperature for 6 hours. After controlling the reaction by LC-MS, 200. mu.L of water was added and the resulting solution was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound E-12 as a white solid (4.6mg, 70%).

m/z(Q-TOF MS ESI+)739.4389(100％,(MH₂)²⁺,C₇₈H₁₁₈N₁₂O₁₆Requirement 739.4389).

Compound E-13

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- (((4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) (methyl) amino) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2- Methylpropanoyl) -L-phenylalanine 2,2, 2-trifluoroacetate salt

Compound E-13-1: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((((4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) (methyl) amino) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine.

Compound E-12-2(185mg,0.123mmol,1.0eq.) was dissolved in a mixture of water (5mL) and acetonitrile (5mL) at room temperature. Piperidine (3.67mL,300eq.) was added and the mixture was stirred at room temperature for 6 hours. The solvent was evaporated to dryness under reduced pressure and the residue was taken up in Et₂O (60mL) grind. Using the solid with Et₂O (20ml) was washed twice with water and dried in vacuo to give Compound E-13-1(175mg, 95%) as an off-white solid.

Compound E-13-2：((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2- ((4- ((((4- ((S) -2-amino-3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) (methyl) amino) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine bis (2,2, 2-trifluoroacetate)

Compound E-13-1(175mg,0.128mmol,1.0eq.) was dissolved in TFA (200. mu.L). After 5 minutes, water (1ml) and acetonitrile (1ml) were added and the solution was lyophilized overnight to give compound E-13-2 as an off-white solid (180mg, 87%).

Compound E-13: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- (((4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) (methyl) amino) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2- Methylpropanoyl) -L-phenylalanine 2,2, 2-trifluoroacetate salt

Compound E-13-2(80mg,0.058mmol,1.0eq.) was dissolved in a mixture of acetonitrile (1.5mL) and DMF (0.4 mL). DIEA (50. mu.L, 0.289mmol,5eq.) was added followed by 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (36mg,0.116mmol,2 eq.). The mixture was stirred at room temperature for 3 hours. After controlling the reaction by LC-MS, the solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC (Waters600E, sunfirep C18 OBD column, 5 μm,19 × 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient 5% to 100% MeCN over 15 min; uv detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound E-13 as a white solid (32mg, 35%).

m/z(Q-TOF MS ESI-)1461.8336(100％,(M-H)^-,C₇₇H₁₁₃N₁₂O₁₆Requirement 1461.8403). m/z (Q-TOF MS ESI +)1463.8565 (2%, MH)⁺,C₇₇H₁₁₅N₁₂O₁₆Requirements 1463.8549),732.4317 (100%, (MH)₂)²⁺,C₇₇H₁₁₆N₁₂O₁₆Requirement 732.4311).

Compound E-15

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3- (((4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) amino) benzyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl Yl) -L-phenylalanine methyl ester 2,2, 2-trifluoroacetate salt

Compound E-15-1: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3- ((((4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) amino) benzyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester 2,2, 2-Trifluoroacetate salt

Compound E-15-1 was prepared according to the same method as compound E-11-6 using carbonate E-11-5(28mg,0.044mmol,1eq.) aniline 15(42mg,0.044mmol,1eq.), HOBt (3mg,0.022mmol,0.5eq.) and DIEA (15 μ L,0.087mmol,2eq.) in DMF (2 mL). Compound E-15-1(8.2mg, 13%) was isolated as a white solid.

Compound E-15-2: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3- ((((4- ((S) -2- ((S) -2-amino-3-methylbutanamido) -5-ureidopentanamido) benzyl) oxy) carbonyl) amino) benzyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester bis (2,2, 2-trifluoroacetic acid salt

Compound E-15-1(8.2mg, 5.58. mu. mol,1.0eq.) was dissolved in TFA (200. mu.L). After 5 minutes, water (1ml) was added and the solution was lyophilized overnight to give compound E-15-8 as a white solid (7.6mg, 99%).

Compound E-15：

Compound E-15 was prepared according to the same method as compound E-12 using amine E-15-2(7.6mg,5.55 μmol,1eq.) 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (2mg,6.65 μmol,1.2eq.) and DIEA (5 μ L,0.028mmol,5eq.) in acetonitrile (0.5 mL). Compound E-15 was isolated as a white solid (4.2mg, 48%).

m/z(Q-TOF MS ESI+)1471.8169(2％,MNa⁺,C₇₆H₁₁₂N₁₂NaO₁₆Requirements 1471.8211),725.4223 (100%, (MH)₂)²⁺,C₇₆H₁₁₄N₁₂O₁₆Requirements 725.4232),483.9482 (10%, (MH)₃)³⁺,C₇₆H₁₁₅N₁₂O₁₆Requirement 483.9513).

Compound F-13

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -N-methyl-5-ureidopentanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-benzenepropanoyl Alanine 2,2, 2-trifluoroacetic acid salt

Compound F-13-1: n- (4- ((tert-Butoxycarbonyl) (methyl) amino) phenethyl) -N-methyl-L-valine benzyl ester

Compound 11C (250mg,0.567mmol,1eq.) was dissolved in THF (10ml) and NaH (60% suspension in mineral oil, 68mg, 1.702mmol,3eq.) was added. The mixture was stirred for 5 min, then iodomethane (106 μ L,1.702mmol,3eq.) was added. The reaction was stirred at room temperature for 2 hours, then quenched with water and separated between EtOAc (100mL) and water (50 mL). The organic phase was washed with MgSO₄Dried and evaporated to dryness to give compound F-13-1(250mg, 97%) as a yellow oil, which was used without further purification.

Compound F-13-2: N-methyl-N- (4- (methylamino) phenethyl) -L-valine benzyl ester

Boc-protected aniline F-13-1(250mg,0.550mmol,1eq) was dissolved in MeOH (5mL) and 1mL of commercially available HCl inⁱSolution in PrOH (5-6M). The solution was stirred at room temperature for 2 hours and then evaporated to dryness under reduced pressure. The resulting yellow oil was used in Et₂Trituration with O to give compound F-13-2 as a yellow solid (202mg, 94%).

Compound F-13-3: n- (4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -N-methyl-5-ureidopentanamido) phenethyl) -N-methyl-L-valine benzyl ester

Acid E-11-3(190mg,0.508mmol,1.5eq.) was dissolved in dry DMF (1ml) and DIEA (118. mu.L, 0.677mmol,2eq.) was added, benzotriazol-1-yl-oxytriazolylphosphonium hexafluorophosphate (PyBOP-264mg,0.508mmol,1.5eq.) and aniline F-13-2(120mg,0.339mmol,1 eq.). The mixture was stirred at room temperature overnight and the solvent was evaporated under reduced pressure. The residue was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound F-13-3 as a white solid (140mg, 45%).

Compound F-13-4: n- (4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -N-methyl-5-ureidopentanamido) phenethyl) -N-methyl-L-valine

Compound F-13-3(116mg,0.163mmol,1eq.) was dissolved in MeOH (5ml) in the presence of Pd/C10% (30mg) and hydrogenated at ambient temperature and atmospheric pressure for 2 hours. The reaction medium is filtered and concentrated under reduced pressure to yield 110mg (99%) of compound F-13-4 as a beige solid.

Compound (I)F-13-5: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -N-methyl-5-ureido-pentanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester 2,2, 2-Trifluoroacetate salt

Amine 3D (89mg,0.140mmol,1eq.) and acid F-13-4(145mg,0.210mmol,1.5eq.) were dissolved in dry DMF (4mL) and PyBOP (109mg,0.210mmol,1.5eq.) and DIEA (73 μ L,0.420mmol,3eq.) were added. The mixture was stirred at room temperature for 1 hour, and the solvent was evaporated. The residue was separated between EtOAc and water and the organic phase was MgSO₄Dried, filtered and evaporated under reduced pressure. The crude product was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound F-13-5 as a white solid (140mg, 73%).

Compound F-13-6: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -N-methyl-5-ureido-pentanoylamino) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine 2, 2-trifluoroacetic acid salt

Compound F-13-5(140mg,0.104mmol,1eq.) was dissolved in a mixture of water (4mL), acetonitrile (4mL) and piperidine (2mL) and stirred at room temperature for 4 hours. The solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound F-13-6(115mg, 83%) as a white solid.

Compound F-13:

compound F-13 was prepared according to the same method as compound E-11 using Boc-protected amine F-13-6(55mg,0.041mmol,1.0eq.) in DCM (0.5mL) and TFA (100 μ L,30eq.), followed by dilution with DMF (1mL), quenching with DIEA (320 μ L,45 eq.), and then reaction with 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (15mg,0.049mmol,1.2 eq.). Compound F-13 was obtained as a white solid (14mg, 24%) after purification by preparative HPLC and lyophilization.

m/z(Q-TOF MS ESI+)1314.8067(2％,MH⁺,C₆₉H₁₀₈N₁₁O₁₄Requirements 1314.8072),657.9067 (100%, (MH)₂)²⁺,C₆₉H₁₀₉N₁₁O₁₄Requirement 657.9072).

Compound F-61

N- ((S) -1- (((S) -1- ((4- ((3R,4S,7S,10S) -4- ((S) -sec-butyl) -7, 10-diisopropyl-3- (2- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 11-dimethyl-6, 9-dioxo-2-oxa-5, 8, 11-triazatridecan-13-yl) phenyl) amino) -1-oxo-5 -ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide 2,2, 2-trifluoroacetate salt

Compound F-61-1: n- (4-Aminophenylethyl) -N-methyl-L-valine benzyl ester dihydrochloride

Compound 11C (1.0g,2.27mmol,1eq.) was dissolved in 8mL of commercially available HClⁱPrOH (5-6M). The mixture was stirred at room temperature for 2 hours and then evaporated to dryness under reduced pressure. The residue was taken up in Et₂O (30ml) was triturated twice and dried in vacuo to give Compound F-61-1(916mg, 98%) as a white solid.

Compound F-61-2: n- (4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) phenethyl) -N-methyl-L-valine benzyl ester

Acid E-11-3(769mg,2.05mmol,1.5eq.) was dissolved in dry DMF (2.5ml) and DIEA (957 μ L,5.48mmol,4eq.) and PyBOP (1.07g,2.05mmol,1.5eq.) were added. Aniline F-61-1(566mg,1.369mmol,1eq.) was added and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the residue was purified on silica gel (DCM/MeOH) to give 969mg (102%) of compound F-61-2 as a white solid.

Compound F-61-3: n- (4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) phenethyl) -N-methyl-L-valine

Compound F-61-2(969mg,1.28mmol,1eq.) was dissolved in MeOH (20ml) in the presence of Pd/C10% (270mg) and hydrogenated at ambient temperature and atmospheric pressure for 3 hours. The reaction medium was filtered and concentrated under reduced pressure, and the residue was purified on silica gel (DCM/MeOH/AcOH) to yield 520mg (67%) of compound F-61-3 as a white solid.

Compound F-61-4: ((S) -1- (((S) -1- ((4- ((3R,4S,7S,10S) -4- ((S) -sec-butyl) -7, 10-diisopropyl-3- (2- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 11-dimethyl-6, 9-dioxo-2-oxa-5, 8, 11-triazatridecan-13-yl) phenyl) amino) -1-oxo-5-ureido Pentane-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamic acid tert-butyl ester 2,2, 2-trifluoroacetate salt

Acid F-61-3(67.5mg,0.111mmol,1.5eq.) was dissolved in dry DMF (2mL) and DECP (17. mu.L, 0.111mmol,1.5eq.) and DIEA (39. mu.L, 0.223mmol,3eq.) were added. After stirring at room temperature for 15 min, amine 1Y (50mg,0.074mmol,1eq.) was added and the solution was stirred overnight. The solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound F61-4(28mg, 28%) as a white solid.

Compound F-61-5: (S) -2- ((S) -2-amino-3-methylbutanamido) -N- (4- ((3R,4S,7S,10S) -4- ((S) -sec-butyl) -7, 10-diisopropyl-3- (2- ((S) -2- ((1R,2R) -1-methoxy-2-methyl-3-oxo-3- (((S) -2-phenyl-1- (thiazol-2-yl) ethyl) amino) propyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 11-dimethyl-6, 9-dioxo-2-oxa-5, 8, 11-Triazatridecan-13-yl) phenyl) -5-ureidovaleramide bis (2,2, 2-trifluoroacetate)

Compound F-61-4(28mg,0.021mmol,1.0eq.) was dissolved in TFA (200 μ L). After 5 minutes, water (2ml) and acetonitrile (0.5ml) were added and the solution was lyophilized overnight to give compound F-61-5(38mg, 134%) as a colorless oil.

Compound F-61：

Compound F-61-5(28.3mg,0.020mmol,1eq.) was dissolved in acetonitrile (0.5mL) and then 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (9mg,0.029 μmol,1.4eq.) and DIEA (25 μ L,0.143mmol,7eq.) were added. The mixture was stirred for 4.5 hours, then HPLC analysis showed the presence of starting material, but the succinimide was completely consumed. Thus, make-up 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (3mg, 0.01. mu. mol,0.5eq.) was added and the reaction was stirred for 1.5 hours. HPLC analysis showed complete consumption of the starting material. The solvent was evaporated to dryness and the residue was taken up in EtOAc/Et₂A mixture of O (80/20) was triturated twice to give Compound F-61 as an off-white solid (19.4mg, 70%).

m/z(Q-TOF MS ESI+)1361.7725(2％,MNa⁺,C₇₀H₁₀₆N₁₂NaO₁₂S requirement 1361.7666),670.3961 (100%, (MH)₂)²⁺,C₇₀H₁₀₈N₁₂O₁₂S requires 670.3960).

Compound F-62：

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester 2,2, 2-Trifluoroacetate salt

Compound F-62-1: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester 2,2, 2-trifluoroacetate.

Compound F-62-1 was prepared in analogy to compound F-61-4 from amine 3D (100mg,0.158mmol,0.9eq.) acid F-61-3(108mg,0.178mmol,1eq.), DECP (41 μ L,0.267mmol,1.5eq.) and DIEA (93 μ L,0.534mmol,3eq.) in DMF (2 mL). After purification by preparative HPLC, Compound F-62-1 was obtained as a white solid (93mg, 39%).

Compound F-62-2: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((S) -2- ((S) -2-amino-3-methylbutanamido) -5-ureidopentanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester bis (2,2, 2-trifluoroacetate)

Compound F-62-1(35mg,0.026mmol,1.0eq.) was dissolved in TFA (200 μ L). After 10 minutes, water (2ml) and acetonitrile (0.5ml) were added and the solution was lyophilized overnight to give compound F-62-2 as a white solid (34mg, 105%).

Compound F-62：

Amine F-62-2(34mg, 5.55. mu. mol,1eq.) was dissolved in acetonitrile (3 mL). DIEA (5. mu.L, 0.028mmol,5eq.) and 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (2mg, 6.65. mu. mol,1.2eq.) were added. HPLC analysis showed complete consumption of the starting material. The solvent was evaporated to dryness and the residue was taken up in EtOAc/Et₂Grinding the mixture of O (80/20). The crude product was purified by preparative HPLC (Waters600E, SunAire PrepC18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound F-62 as a white solid (5.5mg, 13%).

m/z(Q-TOF MS ESI+)1336.7859(2％,MNa⁺,C₆₉H₁₀₇N₁₁NaO₁₄Requirements 1336.7891),657.9073 (100%, (MH)₂)²⁺,C₆₉H₁₀₉N₁₁O₁₄Requirement 657.9072).

Compound F-63：

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine 2,2, 2-Trifluoroacetate salt

Compound F-63-1: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) -3-methylbutanamido) -5-ureidopentanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine

Compound F-62-1(157mg,0.118mmol,1eq.) was dissolved in a mixture of water (4.5mL), acetonitrile (4.5mL) and piperidine (3.5mL) and stirred at room temperature for 5 hours. The solvent was evaporated under reduced pressure and the residue was taken up in Et₂O (60mL) grind. The solid was collected by filtration and taken up in Et₂O (10ml) was washed twice to obtain Compound F-63-1 as an off-white solid (153mg, 100%).

Compound F-63-2: ((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- ((S) -2- ((S) -2-amino-3-methylbutanamido) -5-ureidopentanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine bis 2,2, 2-trifluoroacetate

Compound F-63-1(153mg,0.127mmol,1.0eq.) was dissolved in TFA (200. mu.L). After 10 minutes, water (2ml) and acetonitrile (0.5ml) were added and the solution was lyophilized overnight to give compound F-63-2 as a white solid (34mg, 105%).

Compound F-63：

Amine F-63-2(100mg,0.082mmol,1eq.) was dissolved in a mixture of acetonitrile (2mL) and DMF (0.5mL) and 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (45mg,0.147mmol,1.8eq.) and DIEA (71. mu.L, 0.409mmol,5eq.) were added. After stirring at room temperature for 4.5 hours, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound F-63 as a white solid (42mg, 36%).

m/z(Q-TOF MS ESI+)1300.7901(2％,MH⁺,C₆₈H₁₀₆N₁₁O₁₄Requirements 1300.7915),650.8990 (100%, (MH)₂)²⁺,C₆₈H₁₀₇N₁₁O₁₄Requirement 650.8994).

Compound G-12

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N-methylhexanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester 2,2, 2-trifluoroacetate.

Compound G-12-1: n- (4-Aminophenylethyl) -N-methyl-L-valine benzyl ester dihydrochloride

6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoic acid (200mg,0.947mmol,1eq.) was dissolved in oxalyl chloride (3 mL). The solution was stirred at room temperature for 5 hours and then evaporated to dryness under reduced pressure. Compound G-12-1(217mg, 100%) was obtained as a beige solid and used in the next step without purification.

Compound G-12：

Aniline 12(40mg,0.045mmol,1eq.) was dissolved in dry DCM (1mL) at 0 ℃, and DIEA (8 μ L,0.045mmol,1eq.) was added. After stirring for 30 min, a solution of compound G-12-1(10mg,0.45mmol,1eq.) in dry DCM (1mL) was introduced and the reaction was stirred at 0 ℃ for 1 h. The mixture was diluted with DCM (25mL) and washed twice with water (20mL) and once with brine (10 mL). The organic phase is treated with Na₂SO₄Dried, filtered and evaporated under reduced pressure to give the crude product as a light brown solid (54 mg). It was purified by flash chromatography on silica gel (DCM/MeOH) and then by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). The isolated product was lyophilized to obtain a white solid (23mg), which was repurified by preparative HPLC, and selected fractions were combined and lyophilized to give compound G-12 as a white solid (9mg, 16%).

m/z(Q-TOF MS ESI+)1094.6543(20％,MNa⁺,C₅₉H₈₉N₇NaO₁₁Requirements 1094.6512),1072.6722 (16%, MH)⁺,C₅₉H₉₀N₇O₁₁Requirements 1072.6693),536.8358 (100%, (MH)₂)²⁺,C₅₉H₉₁N₇O₁₁Requirement 536.8383).

Compound G-13

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((4- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N-methylhexanamido) phenethyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine 2,2, 2-trifluoroacetate

Compound G-13：

Aniline 13(15mg,0.015mmol,1eq.) was dissolved in dry DCM (1.5mL) at 0 ℃, and DIEA (8 μ L,0.046mmol,3eq.) was added. A solution of compound G-12-1(3.5mg,0.046mmol,1eq.) in dry DCM (0.5mL) was introduced and the reaction was stirred at 0 ℃ for 1.5 h. The solvent was evaporated under reduced pressure and the crude product was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound G-13 as a white solid (11.4mg, 62%).

m/z(Q-TOF MS ESI+)1058.6510(30％,MH⁺,C₅₈H₈₈N₇O₁₁Requirements 1058.6536),529.8285 (100%, (MH)₂)²⁺,C₅₈H₈₉N₇O₁₁Requirement 529.8305).

Compound G-15

((2R,3R) -3- ((S) -1- ((3R,4S,5S) -4- ((S) -2- ((S) -2- ((3- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) benzyl) (methyl) amino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanoyl) -L-phenylalanine methyl ester 2,2, 2-trifluoroacetate

Compound G-15：

Aniline 15(40mg,0.047mmol,1eq.) was dissolved in dry DCM (2mL) at 0 ℃, and DIEA (10 μ L,0.056mmol,1.2eq.) was added. A solution of compound G-12-1(108mg,0.47mmol,10eq.) in dry DCM (1mL) was introduced and the reaction was stirred at 0 ℃ for 1.5 h. The mixture was diluted with DCM (10mL) and washed twice with water (5 mL). The organic phase was washed with MgSO₄Dried, filtered and evaporated under reduced pressure to give the crude product as a beige solid. It was purified by preparative HPLC (Waters600E, SunAire Prep C18 OBD column, 5 μm, 19X 100 mm; mobile phase: 0.1% TFA buffered water/MeCN; gradient from 5% to 100% MeCN over 15 min; UV detector Waters 2487 at 220 nm). Selected fractions were combined and lyophilized to give compound G15 as a white solid (27mg, 50%).

m/z(Q-TOF MS ESI+)1066.6517(2％,MNa⁺,C₅₇H₈₅N₇NaO₁₁Requirements 1066.6199),522.8224 (100%, (MH)₂)²⁺,C₅₇H₈₇N₇O₁₁Requirement 522.8226).

Example 17: synthesis, purification and characterization of ADCs

The procedure described below applies to both the chimeric IgG1 format and the humanized IgG1 format. It must be understood that for any other format, such as IgG2, IgG4, etc., one skilled in the art can use general knowledge to alter the procedure.

Antibodies (1-5mg/ml) were partially reduced for 2h at 37 ℃ with tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in 10mM borate buffer (pH 8.4) containing 150mM NaCl and 2mM EDTA. Typically, 2.5-3 molar equivalents of TCEP are used to target a drug-antibody ratio (DAR) of about 4, respectively. Under non-reducing conditions, partial antibody reduction was confirmed by SDS-PAGE analysis. The reduction mixture was allowed to cool to room temperature before coupling the linker-drug to the released interchain cysteine residues. Then, the antibody concentration was adjusted to 1mg/ml with 10mM borate buffer (pH 8.4) containing 150mM NaCl and 2mM EDTA, and 5 molar excess of the drug as compared to the antibody was added from 10mM dimethyl sulfoxide (DMSO) solution. The final DMSO concentration was adjusted to 10% to maintain the solubility of the drug in aqueous media during coupling. The reaction was carried out at room temperature for 1 h. Drug overdose was quenched by addition of 1.5 moles of N-acetylcysteine per mole of drug and incubation at room temperature for 1 hour. After dialysis overnight at 4 ℃ against 25mM His buffer (pH 6.5) containing 150mM NaCl, the antibody-drug-conjugate was purified by using methods known to those skilled in the art, which are based on commercially available chromatography columns and ultrafiltration units. First, unconjugated drug and ADC aggregates were removed by Size Exclusion Chromatography (SEC) on S200(GE Life Sciences) or TSK G3000SW (Tosoh) columns. The purified ADC monomers were then concentrated to 2-3mg/ml by ultrafiltration on a 30 or 50kDa MWCO filtration unit, or by affinity chromatography on protein a. After sterile filtration on a 0.2 μm filter, the purified ADC was stored at 4 ℃. They were further analyzed by SDS-PAGE under reducing and non-reducing conditions to confirm drug conjugation and by SEC on analytical S200 or TSK G3000SWXL columns to determine the content of monomeric and aggregated forms. Protein concentration was determined by using a bicinchoninic acid (BCA) assay with IgG as standard. The DAR for each purified ADC was estimated by HIC and LC-MS. Typically, the content of aggregated forms is below 5% and DAR is between 3.5 and 5.

Example 18: evaluation of cytotoxicity of IGF-1R antibodies conjugated to different drugs

18.1 evaluation of chimeric antibodies in MCF-7 cells

Five IGF-1R antibodies have been shown to be rapidly internalized into lysosomes and have low binding capacity in acidic environments. In this regard, those abs have all the properties to function as ADCs. Thus, five chimeric anti-IGF-1R antibodies were conjugated to three different compounds (G-13; E-13 and F-63). The drug-antibody ratio of these ADCs is about 4. To assess non-specific cytotoxicity, an irrelevant chimeric antibody c9G4 was also coupled with those compounds with the same DAR. In complete culture medium, MCF-7 cells and increasing concentrations of each ADC at 37 degrees C were incubated for 6 days. Cell viability was assessed using a fluorescent cell viability assay (CellTiter-Glo, Promega). The fluorescence signal was read using a Mithras plate reader (Berthold Technologies). An unrelated chimeric antibody, c9G4, conjugated to E-13, G-13 or F-63, showed no or mild cytotoxic activity in MCF-7 cells (FIG. 21). In contrast, the addition of all other ADCs obtained after coupling the anti-IGF-1R antibody to E-13, G-13 or F-63 significantly reduced MCF-7 cell viability.

18.2 evaluation of chimeric antibodies in Normal cells

The expression level of IGF-1R was evaluated in primary normal cells (Promocell GmbH) using c208F2 mAb. For this purpose, cells (0.5X 10) were plated in FACS buffer (PBS, 0.1% BSA, 0.01% NaN3)⁶Individual cells/ml) was incubated with 10. mu.g/ml of c208F2 antibody at 4 ℃ for 20 min. They were then washed 3 times and incubated with the appropriate secondary antibody conjugated to Alexa488 at 4 ℃ for an additional 20 minutes in the dark, followed by 3 washes in FACS buffer. Propidium iodide (which stains dead cells) is used to identify viable cells in which binding of anti-IGF-1R antibodies is immediately performed. Expression levels (Bmax) in normal cells were low compared to IGF-1R expression in MCF-7 cells (see example 2, Table 8) (Table 14).

TABLE 14

Normal cells	Bmax
		Human aortic endothelial cell (HAoEC)	21
Human lung microvascular endothelial cells (HPMEC)	33
		Human Bronchial Smooth Muscle Cells (HBSMC)	26
Human renal epithelial cells (HREpC)	110
		Human urothelial cell (HUG)	181

ADC c208F2-G-13 was evaluated for cytotoxicity in normal cells. In complete culture medium, cells with increased concentrations of c208F2-G-13 at 37 degrees C were incubated for 6 days. Cell viability was assessed using a fluorescent cell viability assay (CellTiter-Glo, Promega). The fluorescence signal was read using a Mithras plate reader (Berthold Technologies). In HBSMC, HPMEC, HAoEC and HREpC, no major cytotoxicity was observed (FIG. 25). In HUC, less cytotoxicity was measured only at high concentrations of c208F 2-G-13.

18.3 evaluation of humanized variants of hz208F2

Sixteen humanized variants of 208F2 were conjugated to compound G-13. The drug-antibody ratio of these ADCs is about 4. To assess non-specific cytotoxicity, an irrelevant chimeric antibody c9G4 was also coupled with those compounds with the same DAR. Chimeric antibody c208F2 was also coupled to G-13. In complete culture medium, MCF-7 cells and increasing concentrations of each ADC at 37 degrees C were incubated for 6 days. Cell viability was assessed using a fluorescent cell viability assay (CellTiter-Glo, Promega). The fluorescence signal was read using a Mithras plate reader (Berthold Technologies). The G-13-conjugated unrelated chimeric antibody c9G4 showed no or mild cytotoxic activity in MCF-7 cells (FIG. 26). In contrast, the addition of all other ADCs obtained after coupling the anti-IGF-1R antibody to G-13 significantly reduced MCF-7 cell viability. The ability of the sixteen humanized variants to induce cytotoxicity was at least equal to or even better than that measured with the chimeric form c208F2-G-13, as shown in table 15, and as illustrated with one humanized variant in fig. 26.

Watch 15

Example 19: c208F2 anti-antibody conjugated with E-13, G-13 or F-63 compounds in MCF-7 xenograft models In vivo activity of the body.

To confirm that the in vitro efficacy of c208F2 conjugated to G-13, E-13 or F-63 compounds can be transformed in vivo, they have been tested in the MCF-7 xenograft model.

All animal procedures were performed according to the 2010/63/UE Directive (Directive) guidelines on protecting animals for scientific purposes. The protocol was approved by the Animal ethics committee of the Pierre Fabre Institute (Animal ethical committee). Five million MCF-7 cells were injected subcutaneously into 7 week old Swiss/Nude mice. Prior to cell injection, estrogen granules (pellets) (Innovative Research of America) were implanted in the left flank of the mice to release the estrogen necessary for the in vivo growth of MCF-7 tumors.

Twenty days after MCF-7 cell implantation, when the tumor reaches 120-150mm³At average size of (a), animals were grouped according to tumor size and appearance, with 5 mice per group. Different treatments were inoculated by intraperitoneal injection. The health of the animals was monitored daily. Tumor volumes were measured twice weekly with electronic calipers until the end of the study. Tumor volume was calculated using the formula: π/6 × Length × Width × height. Toxicity was evaluated after weighing the animals three times a week. Statistical analysis was performed at each measurement using the Mann-Whitney test. All compounds were injected intraperitoneally (i.p.). In this example, the anti-tumor activity of C208F2 mAb conjugated to E-13, F-13 or F-63 at approximately DAR 4 was evaluated after 7mg/kg doses of D20 and D272 (FIGS. 22A, 22B and 22C). In parallel, capped (capped) drug moieties E-13, F-13 and F-63 were injected at equivalent doses corresponding to doses of c208F2-E-13, c208F2-F-13 and c208F2-F-63 of about 4 DAR at 7 mg/kg.

Injection of C208-E-13 (fig. 22A), C208F2-G-13 (fig. 22B), or C208F2-F-63 (fig. 22C) significantly inhibited and even induced complete tumor growth regression (p <0.05 vs. corresponding capped drugs). No statistical activity differences were noted between c208-E-13, c208F2-G-13, and c208F 2-F-63. The capped drug had no effect on MCF-7 tumor growth (p >0.05 vs control).

A second set of experiments was performed in the MCF-7 xenograft model using c208F2 conjugated to E-13 or G-13 and an unrelated antibody c9G4 conjugated to E-13 or G-13, as described previously. Mice were i.p. injected with 7mg/kg of each ADC at D20 and D27 (fig. 23A and 23B).

Injection of c9G4-E-13 and c9G4-F-13 gently and transiently affected the growth of MCF-7 xenograft tumors. However, this second experiment confirmed that injection of c208-E-13 or c208F2-G-13 induced complete tumor regression, as D43 showed high anti-tumor activity of these ADCs.

⁺Examples20: in vivo of the hz208F2 antibody conjugated to a G-13 Compound in a 3MCF-7 xenograft model And (4) activity.

In the MCF-7 xenograft model, a humanized form of 208F2 coupled to a G-13 compound has been evaluated in vivo.

All animal procedures were performed according to the 2010/63/UE Directive (Directive) guidelines on protecting animals for scientific purposes. The protocol was approved by the animal ethics committee of the Pierre Fabre Institute. Five million MCF-7 cells were injected subcutaneously into 7 week old Swiss/Nude mice. Prior to cell injection, estrogen particles (innovative research of America) were implanted in the left flank of the mice to release the estrogen necessary for the in vivo growth of the MCF-7 tumor.

Twenty days after MCF-7 cell implantation, when the tumor reaches 120-150mm³At average size of (a), animals were grouped according to tumor size and appearance, with 6 mice per group. Different treatments were inoculated by intraperitoneal injection as a 4-injection schedule; injections were given every four days (Q4d 4). The health of the animals was monitored daily. Tumor volumes were measured twice weekly with electronic calipers until the end of the study. Tumor volume was calculated using the formula: π/6 × Length × Width × height. Toxicity was evaluated after weighing the animals three times a week. Statistical analysis was performed at each measurement using the Mann-Whitney test. All compounds were injected intraperitoneally (i.p.). In this example, the anti-tumor activity of c208F2 mAb conjugated to G-13 compound was compared to a different humanized form also conjugated to G-13 (fig. 27). The humanized forms tested are described in table 16 below:

TABLE 16

Injection of the humanized form of c208-G-13 or 208F2 significantly inhibited, even induced complete regression of tumor growth (p <0.05 vs. corresponding controls). No statistical activity difference was observed between c208F2-G-13 and the humanized forms tested.

As described previously, a second set of experiments were performed in the MCF-7 xenograft model using either c208F2 or hz208F2-4 coupled to G-13 (FIGS. 28A and 28B, respectively). Mice were injected i.p. with 3mg/kg of each ADC, 4 injections every four days (Q4d4) or only once.

In the MCF xenograft model, the same strong antitumor activity was observed when ADC was injected four times or only once.

⁺Example 21: preparation of 208F2 antibody conjugated with G-13 or E-13 Compound in 2CaOV-3 xenograft model And (4) in vivo activity.

Is also at 2⁺Expression tumors, antitumor activity was studied in a CaOV-3 xenograft model, which is an ovarian cancer cell line. For this program, mice were injected subcutaneously with 7X 10 at D0⁶And (4) cells. When the tumor reaches about 120mm³(19 days after tumor cell injection), the animals were divided into 5 groups of 5 mice with comparable tumor size, each group of 5 mice, and treated intraperitoneally with c208F2 conjugated to E-13 or G-13 and an irrelevant antibody c9G4 conjugated to E-13 or G-13. Mice were i.p. injected with 3mg/kg of each ADC for 6 injection cycles; once every four days. The xenograft growth rate of mice was followed. Tumor volume was calculated by the following formula: π/6 × Length × Width × height.

Injection of c9G4-G-13 did not affect the growth of CaOV-3 xenograft tumors compared to c9G4-E-13, which gently and transiently induced growth retardation. Meanwhile, injection of c208F2-E-13 or c208F2-G-13 induced 95% and 77% tumor growth inhibition, respectively, on day 50 (FIGS. 29A and 29B).

Sequence listing

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<120> IGF-1R antibody-drug-conjugates and their use for the treatment of cancer

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Gln Gln Gly Ser Thr Leu Pro Tyr Thr

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Gln Asp Ile Asn Lys Tyr

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Gln Gln Gly Ser Thr Leu Pro Tyr Thr

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Gln Gln Gly Ser Ala Leu Pro Tyr Thr

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

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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser

115 120

<210>14

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<223> c212A11, heavy chain, VH

<400>14

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser

115 120

<210>15

<211>120

<212>PRT

<213> Artificial sequence

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<223> c214F8, heavy chain, VH

<400>15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

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Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser

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<210>16

<211>120

<212>PRT

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<223> c219D6, heavy chain, VH

<400>16

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Gly LysThr Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Phe Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser

115 120

<210>17

<211>120

<212>PRT

<213> Artificial sequence

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<223> c213B10, heavy chain, VH

<400>17

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 4045

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser

115 120

<210>18

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<212>PRT

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<223> c208F2, light chain, VL

<400>18

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

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Ile Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>19

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<223> c212A11, light chain, VL

<400>19

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

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>20

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<223> c214F8, light chain, VL

<400>20

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

1 5 10 15

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

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Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Ile Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>21

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<223> c219D6, light chain, VL

<400>21

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>22

<211>107

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<223> c213B10, light chain, VL

<400>22

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Ile Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>23

<211>449

<212>PRT

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<223> c208F2, heavy chain, full length

<400>23

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>24

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> c212A11, heavy chain, full length

<400>24

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>25

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> c214F8, heavy chain, full length

<400>25

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>26

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> c219D6, heavy chain, full Length

<400>26

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Phe Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>27

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> c213B10, heavy chain, full length

<400>27

Gln Val Gln Leu Gln Gln Ser Gly Ser GluLeu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>28

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> c208F2, light chain, full length

<400>28

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Ile Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 7580

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>29

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> c212A11, light chain, full length

<400>29

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>30

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> c214F8, light chain, full length

<400>30

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Ile Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>31

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> c219D6, light chain, full Length

<400>31

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>32

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> c213B10, light chain, full length

<400>32

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Gln Pro Asp Gly Thr Ile Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys AlaAsp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>33

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 (var.1) heavy chain, VH

<400>33

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Trp Pro Gly Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>34

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223>hz208F2 (var. 3), VH

<400>34

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 4045

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

50 55 60

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

65 70 75 80

Met Phe Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>35

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>hz208F2 (var. 1), VL

<400>35

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>36

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>hz208F2 (var.3), VL

<400>36

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Arg Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>37

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 (var.1), heavy chain, full length

<400>37

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

1 5 1015

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Trp Pro Gly Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>38

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 (var.3), full length of heavy chain

<400>38

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Phe Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>39

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 (var.1), light chain, full length

<400>39

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>40

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 (var.3), light chain, full length

<400>40

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Arg Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>41

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 (var.2) heavy chain, VH

<220>

<221>MISC_FEATURE

<222>(20)..(20)

<223> Met may be replaced by Val

<220>

<221>MISC_FEATURE

<222>(34)..(34)

<223> Ile can be replaced by Met

<220>

<221>MISC_FEATURE

<222>(35)..(35)

<223> Tyr can be replaced by His

<220>

<221>MISC_FEATURE

<222>(38)..(38)

<223> Lys may be replaced by Arg

<220>

<221>MISC_FEATURE

<222>(48)..(48)

<223> Leu may be replaced by Met

<220>

<221>MISC_FEATURE

<222>(50)..(50)

<223> Trp can be replaced by Ile

<220>

<221>MISC_FEATURE

<222>(59)..(59)

<223> Lys may be replaced by Ser

<220>

<221>MISC_FEATURE

<222>(61)..(61)

<223> Asn can be replaced by Ala

<220>

<221>MISC_FEATURE

<222>(62)..(62)

<223> Glu may be replaced by Gln

<220>

<221>MISC_FEATURE

<222>(70)..(70)

<223> Leu may be replaced by Met

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> Ala may be replaced by Arg

<220>

<221>MISC_FEATURE

<222>(74)..(74)

<223> Lys may be replaced by Thr

<220>

<221>MISC_FEATURE

<222>(76)..(76)

<223> Ser may be replaced by Thr

<220>

<221>MISC_FEATURE

<222>(77)..(77)

<223> Asn can be replaced by Ser

<220>

<221>MISC_FEATURE

<222>(79)..(79)

<223> Ala may be replaced by Val

<220>

<221>MISC_FEATURE

<222>(82)..(82)

<223> Phe can be replaced by Glu

<220>

<221>MISC_FEATURE

<222>(95)..(95)

<223> Phe can be replaced by Tyr

<400>41

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Met Phe Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>42

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 (var.2), light chain, VL

<220>

<221>MISC_FEATURE

<222>(22)..(22)

<223> Ser may be replaced by Thr

<220>

<221>MISC_FEATURE

<222>(53)..(53)

<223> Arg may be replaced by Ser

<220>

<221>MISC_FEATURE

<222>(55)..(55)

<223> His can be replaced by Gln

<220>

<221>MISC_FEATURE

<222>(65)..(65)

<223> Arg may be replaced by Ser

<220>

<221>MISC_FEATURE

<222>(71)..(71)

<223> Tyr can be replaced by Phe

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> Ser may be replaced by Thr

<220>

<221>MISC_FEATURE

<222>(77)..(77)

<223> Asn can be replaced by Ser

<220>

<221>MISC_FEATURE

<222>(87)..(87)

<223> Phe can be replaced by Tyr

<400>42

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Arg Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>43

<211>329

<212>PRT

<213> Artificial sequence

<220>

<223> constant Domain (VH) IgG1

<400>43

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

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

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

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

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210>44

<211>326

<212>PRT

<213> Artificial sequence

<220>

<223> constant Domain (VH) IgG4 (S228P)

<400>44

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

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

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210>45

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> kappa domain (VL)

<400>45

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

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

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>46

<211>98

<212>PRT

<213> Artificial sequence

<220>

<223> human germline IGHV1-46 x 01

<400>46

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg

<210>47

<211>95

<212>PRT

<213> Artificial sequence

<220>

<223> human germline IGKV1-39 x 01

<400>47

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

1 510 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

<210>48

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> human germline IGHJ4 x 01

<400>48

Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10 15

<210>49

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> human germline IGKJ4 x 01

<400>49

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

1 5 10

<210>50

<211>1367

<212>PRT

<213> Artificial sequence

<220>

<223> IGF-1R (human)

<400>50

Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu

1 5 10 15

Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu Ile

20 25 30

Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu Lys Arg

35 40 45

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

5055 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Asn Tyr Ala Leu Val Ile Phe Glu Met Thr Asn Leu Lys Asp Ile

115 120 125

Gly Leu Tyr Asn Leu Arg Asn Ile Thr Arg Gly Ala Ile Arg Ile Glu

130 135 140

Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu Ile

145 150 155 160

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

165 170 175

Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys

180 185 190

Glu Lys Thr Thr Ile Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr

195 200 205

Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys

210 215 220

Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser

225 230 235 240

Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr

245 250 255

Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu

260 265 270

Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu Ser Ala

275 280 285

Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp Gly Glu Cys Met

290 295 300

Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser Met Tyr

305 310 315 320

Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys

325 330 335

Lys Thr Lys ThrIle Asp Ser Val Thr Ser Ala Gln Met Leu Gln Gly

340 345 350

Cys Thr Ile Phe Lys Gly Asn Leu Leu Ile Asn Ile Arg Arg Gly Asn

355 360 365

Asn Ile Ala Ser Glu Leu Glu Asn Phe Met Gly Leu Ile Glu Val Val

370 375 380

Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val Ser Leu Ser

385 390 395 400

Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu Gln Leu Glu Gly

405 410 415

Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln Leu Trp

420 425 430

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

435 440 445

Ala Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr Arg Met Glu Glu

450 455 460

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

465 470 475 480

Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Leu His Phe Thr

485 490 495

Ser Thr Thr Thr Ser Lys Asn Arg Ile Ile Ile Thr Trp His Arg Tyr

500 505 510

Arg Pro Pro Asp Tyr Arg Asp Leu Ile Ser Phe Thr Val Tyr Tyr Lys

515 520 525

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

530 535 540

Gly Ser Asn Ser Trp Asn Met Val Asp Val Asp Leu Pro Pro Asn Lys

545 550 555 560

Asp Val Glu Pro Gly Ile Leu Leu His Gly Leu Lys Pro Trp Thr Gln

565 570 575

Tyr Ala Val Tyr Val Lys Ala Val Thr Leu Thr Met Val Glu Asn Asp

580 585 590

His Ile Arg Gly Ala Lys Ser Glu Ile Leu Tyr Ile Arg Thr Asn Ala

595 600 605

Ser Val Pro Ser Ile Pro Leu Asp Val Leu Ser Ala Ser Asn Ser Ser

610615 620

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

625 630 635 640

Leu Ser Tyr Tyr Ile Val Arg Trp Gln Arg Gln Pro Gln Asp Gly Tyr

645 650 655

Leu Tyr Arg His Asn Tyr Cys Ser Lys Asp Lys Ile Pro Ile Arg Lys

660 665 670

Tyr Ala Asp Gly Thr Ile Asp Ile Glu Glu Val Thr Glu Asn Pro Lys

675 680 685

Thr Glu Val Cys Gly Gly Glu Lys Gly Pro Cys Cys Ala Cys Pro Lys

690 695 700

Thr Glu Ala Glu Lys Gln Ala Glu Lys Glu Glu Ala Glu Tyr Arg Lys

705 710 715 720

Val Phe Glu Asn Phe Leu His Asn Ser Ile Phe Val Pro Arg Pro Glu

725 730 735

Arg Lys Arg Arg Asp Val Met Gln Val Ala Asn Thr Thr Met Ser Ser

740 745 750

Arg Ser Arg Asn Thr Thr Ala Ala Asp Thr Tyr Asn Ile Thr Asp Pro

755 760 765

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

770 775 780

Lys Glu Arg Thr Val Ile Ser Asn Leu Arg Pro Phe Thr Leu Tyr Arg

785 790 795 800

Ile Asp Ile His Ser Cys Asn His Glu Ala Glu Lys Leu Gly Cys Ser

805 810 815

Ala Ser Asn Phe Val Phe Ala Arg Thr Met Pro Ala Glu Gly Ala Asp

820 825 830

Asp Ile Pro Gly Pro Val Thr Trp Glu Pro Arg Pro Glu Asn Ser Ile

835 840 845

Phe Leu Lys Trp Pro Glu Pro Glu Asn Pro Asn Gly Leu Ile Leu Met

850 855 860

Tyr Glu Ile Lys Tyr Gly Ser Gln Val Glu Asp Gln Arg Glu Cys Val

865 870 875 880

Ser Arg Gln Glu Tyr Arg Lys Tyr Gly Gly Ala Lys Leu Asn Arg Leu

885 890 895

Asn Pro Gly Asn Tyr Thr Ala Arg Ile Gln Ala Thr Ser Leu Ser Gly

900 905 910

Asn Gly Ser Trp Thr Asp Pro Val Phe Phe Tyr Val Gln Ala Lys Thr

915 920 925

Gly Tyr Glu Asn Phe Ile His Leu Ile Ile Ala Leu Pro Val Ala Val

930 935 940

Leu Leu Ile Val Gly Gly Leu Val Ile Met Leu Tyr Val Phe His Arg

945 950 955 960

Lys Arg Asn Asn Ser Arg Leu Gly Asn Gly Val Leu Tyr Ala Ser Val

965 970 975

Asn Pro Glu Tyr Phe Ser Ala Ala Asp Val Tyr Val Pro Asp Glu Trp

980 985 990

Glu Val Ala Arg Glu Lys Ile Thr Met Ser Arg Glu Leu Gly Gln Gly

995 1000 1005

Ser Phe Gly Met Val Tyr Glu Gly Val Ala Lys Gly Val Val Lys

1010 1015 1020

Asp Glu Pro Glu Thr Arg Val Ala Ile Lys Thr Val Asn Glu Ala

1025 1030 1035

Ala Ser Met Arg Glu Arg Ile Glu Phe Leu Asn Glu Ala Ser Val

1040 1045 1050

Met Lys Glu Phe Asn Cys His His Val Val Arg Leu Leu Gly Val

1055 1060 1065

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

1070 1075 1080

Arg Gly Asp Leu Lys Ser Tyr Leu Arg Ser Leu Arg Pro Glu Met

1085 1090 1095

Glu Asn Asn Pro Val Leu Ala Pro Pro Ser Leu Ser Lys Met Ile

1100 1105 1110

Gln Met Ala Gly Glu Ile Ala Asp Gly Met Ala Tyr Leu Asn Ala

1115 1120 1125

Asn Lys Phe Val His Arg Asp Leu Ala Ala Arg Asn Cys Met Val

1130 1135 1140

Ala Glu Asp Phe Thr Val Lys Ile Gly Asp Phe Gly Met Thr Arg

1145 1150 1155

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

1160 1165 1170

Leu Pro Val Arg Trp Met Ser Pro Glu Ser Leu Lys Asp Gly Val

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

Glu Gln Val Leu Arg Phe Val Met Glu Gly Gly Leu Leu Asp Lys

1220 1225 1230

Pro Asp Asn Cys Pro Asp Met Leu Phe Glu Leu Met Arg Met Cys

1235 1240 1245

Trp Gln Tyr Asn Pro Lys Met Arg Pro Ser Phe Leu Glu Ile Ile

1250 1255 1260

Ser Ser Ile Lys Glu Glu Met Glu Pro Gly Phe Arg Glu Val Ser

1265 1270 1275

Phe Tyr Tyr Ser Glu Glu Asn Lys Leu Pro Glu Pro Glu Glu Leu

1280 1285 1290

Asp Leu Glu Pro Glu Asn Met Glu Ser Val Pro Leu Asp Pro Ser

1295 1300 1305

Ala Ser Ser Ser Ser Leu Pro Leu Pro Asp Arg His Ser Gly His

1310 1315 1320

Lys Ala Glu Asn Gly Pro Gly Pro Gly Val Leu Val Leu Arg Ala

1325 1330 1335

Ser Phe Asp Glu Arg Gln Pro Tyr Ala His Met Asn Gly Gly Arg

1340 1345 1350

Lys Asn Glu Arg Ala Leu Pro Leu Pro Gln Ser Ser Thr Cys

1355 1360 1365

<210>51

<211>932

<212>PRT

<213> Artificial sequence

<220>

<223> IGF-1R ECD (human)

<400>51

Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu

1 5 10 15

Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu Ile

20 25 30

Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu Lys Arg

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Asn Tyr Ala Leu Val Ile Phe Glu Met Thr Asn Leu Lys Asp Ile

115 120 125

Gly Leu Tyr Asn Leu Arg Asn Ile Thr Arg Gly Ala Ile Arg Ile Glu

130 135 140

Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu Ile

145 150 155 160

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

165 170 175

Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys

180 185190

Glu Lys Thr Thr Ile Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr

195 200 205

Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys

210 215 220

Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser

225 230 235 240

Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr

245 250 255

Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu

260 265 270

Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu Ser Ala

275 280 285

Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp Gly Glu Cys Met

290 295 300

Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser Met Tyr

305 310 315 320

Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys

325 330 335

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

340 345 350

Cys Thr Ile Phe Lys Gly Asn Leu Leu Ile Asn Ile Arg Arg Gly Asn

355 360 365

Asn Ile Ala Ser Glu Leu Glu Asn Phe Met Gly Leu Ile Glu Val Val

370 375 380

Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val Ser Leu Ser

385 390 395 400

Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu Gln Leu Glu Gly

405 410 415

Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln Leu Trp

420 425 430

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

435 440 445

Ala Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr Arg Met Glu Glu

450 455 460

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

465 470 475 480

Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Leu His Phe Thr

485 490 495

Ser Thr Thr Thr Ser Lys Asn Arg Ile Ile Ile Thr Trp His Arg Tyr

500 505 510

Arg Pro Pro Asp Tyr Arg Asp Leu Ile Ser Phe Thr Val Tyr Tyr Lys

515 520 525

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

530 535 540

Gly Ser Asn Ser Trp Asn Met Val Asp Val Asp Leu Pro Pro Asn Lys

545 550 555 560

Asp Val Glu Pro Gly Ile Leu Leu His Gly Leu Lys Pro Trp Thr Gln

565 570 575

Tyr Ala Val Tyr Val Lys Ala Val Thr Leu Thr Met Val Glu Asn Asp

580 585 590

His Ile Arg Gly Ala Lys Ser Glu Ile Leu Tyr Ile Arg Thr Asn Ala

595 600 605

Ser Val Pro Ser Ile Pro Leu Asp Val Leu Ser Ala Ser Asn Ser Ser

610 615 620

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

625 630 635 640

Leu Ser Tyr Tyr Ile Val Arg Trp Gln Arg Gln Pro Gln Asp Gly Tyr

645 650 655

Leu Tyr Arg His Asn Tyr Cys Ser Lys Asp Lys Ile Pro Ile Arg Lys

660 665 670

Tyr Ala Asp Gly Thr Ile Asp Ile Glu Glu Val Thr Glu Asn Pro Lys

675 680 685

Thr Glu Val Cys Gly Gly Glu Lys Gly Pro Cys Cys Ala Cys Pro Lys

690 695 700

Thr Glu Ala Glu Lys Gln Ala Glu Lys Glu Glu Ala Glu Tyr Arg Lys

705 710 715 720

Val Phe Glu Asn Phe Leu His Asn Ser Ile Phe Val Pro Arg Pro Glu

725 730 735

Arg Lys Arg Arg Asp Val Met Gln Val Ala Asn Thr Thr Met Ser Ser

740 745750

Arg Ser Arg Asn Thr Thr Ala Ala Asp Thr Tyr Asn Ile Thr Asp Pro

755 760 765

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

770 775 780

Lys Glu Arg Thr Val Ile Ser Asn Leu Arg Pro Phe Thr Leu Tyr Arg

785 790 795 800

Ile Asp Ile His Ser Cys Asn His Glu Ala Glu Lys Leu Gly Cys Ser

805 810 815

Ala Ser Asn Phe Val Phe Ala Arg Thr Met Pro Ala Glu Gly Ala Asp

820 825 830

Asp Ile Pro Gly Pro Val Thr Trp Glu Pro Arg Pro Glu Asn Ser Ile

835 840 845

Phe Leu Lys Trp Pro Glu Pro Glu Asn Pro Asn Gly Leu Ile Leu Met

850 855 860

Tyr Glu Ile Lys Tyr Gly Ser Gln Val Glu Asp Gln Arg Glu Cys Val

865 870 875 880

Ser Arg Gln Glu Tyr Arg Lys Tyr Gly Gly Ala Lys Leu Asn Arg Leu

885 890 895

Asn Pro Gly Asn Tyr Thr Ala Arg Ile Gln Ala Thr Ser Leu Ser Gly

900 905 910

Asn Gly Ser Trp Thr Asp Pro Val Phe Phe Tyr Val Gln Ala Lys Thr

915 920 925

Gly Tyr Glu Asn

930

<210>52

<211>512

<212>PRT

<213> Artificial sequence

<220>

<223> IGF-1R ECD N-terminal (human)

<400>52

Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu

1 5 10 15

Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu Ile

20 25 30

Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu Lys Arg

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Asn Tyr Ala Leu Val Ile Phe Glu Met Thr Asn Leu Lys Asp Ile

115 120 125

Gly Leu Tyr Asn Leu Arg Asn Ile Thr Arg Gly Ala Ile Arg Ile Glu

130 135 140

Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu Ile

145 150 155 160

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

165 170 175

Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys

180 185 190

Glu Lys Thr Thr Ile Asn AsnGlu Tyr Asn Tyr Arg Cys Trp Thr Thr

195 200 205

Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys

210 215 220

Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser

225 230 235 240

Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr

245 250 255

Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu

260 265 270

Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu Ser Ala

275 280 285

Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp Gly Glu Cys Met

290 295 300

Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser Met Tyr

305 310 315 320

Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys

325 330 335

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

340 345 350

Cys Thr Ile Phe Lys Gly Asn Leu Leu Ile Asn Ile Arg Arg Gly Asn

355 360 365

Asn Ile Ala Ser Glu Leu Glu Asn Phe Met Gly Leu Ile Glu Val Val

370 375 380

Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val Ser Leu Ser

385 390 395 400

Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu Gln Leu Glu Gly

405 410 415

Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln Leu Trp

420 425 430

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

435 440 445

Ala Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr Arg Met Glu Glu

450 455 460

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

465 470475 480

Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Leu His Phe Thr

485 490 495

Ser Thr Thr Thr Ser Lys Asn Arg Ile Ile Ile Thr Trp His Arg Tyr

500 505 510

<210>53

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> tetrapeptide (linker)

<400>53

Gly Phe Leu Gly

1

<210>54

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> tetrapeptide (linker)

<400>54

Ala Leu Ala Leu

1

<210>55

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> tetrapeptide (linker)

<400>55

Pro Val Gly Val Val

1 5

<210>56

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H037, VH

<400>56

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>57

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 light chain L018, VL

<400>57

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Arg Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>58

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H037 full length

<400>58

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 4045

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>59

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 light chain L018 full Length

<400>59

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Arg Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>60

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 light chain L021, VL

<400>60

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>61

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 light chain L021 full length

<400>61

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>62

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H047, VH

<400>62

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>63

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H047 full length

<400>63

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>64

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H049, VH

<400>64

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>65

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H049 full length

<400>65

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>66

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H051, VH

<400>66

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>67

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H051 full length

<400>67

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser ValPhe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>68

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H052, VH

<400>68

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>69

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H052 full length

<400>69

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

1 5 1015

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>70

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H057, VH

<400>70

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Lys Ser Thr Asn Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>71

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H057 full length

<400>71

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Lys Ser Thr Asn Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>72

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H068, VH

<400>72

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>73

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H068 full length

<400>73

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>74

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H070, VH

<400>74

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>75

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H070 full length

<400>75

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr PhePro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440445

Gly

<210>76

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H071, VH

<400>76

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>77

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H071 full length

<400>77

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

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

65 7075 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>78

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H076, VH

<400>78

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>79

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H076 full length

<400>79

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210>80

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H077, VH

<400>80

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

5055 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>81

<211>449

<212>PRT

<213> Artificial sequence

<220>

<223> hz208F2 heavy chain H077 full length

<400>81

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Trp Pro Gly Asp Gly Ser Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Met Ile Thr Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

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

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

Claims

1. An antibody-drug conjugate of the following formula (I):

Ab-(L-D)_n

(I)

or a pharmaceutically acceptable salt thereof,

wherein

Ab is an antibody or antigen-binding fragment thereof capable of binding to human IGF-1R, said Ab being selected from

a) An antibody comprising heavy chain CDR1, CDR2 and CDR3 having the sequences SEQ ID nos. 7,2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 having the sequences SEQ ID nos. 9, 5 and 11, respectively;

b) an antibody comprising heavy chain CDR1, CDR2 and CDR3 having the sequences SEQ ID nos. 7,2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 having the sequences SEQ ID nos. 10, 5 and 11, respectively;

c) an antibody comprising heavy chain CDR1, CDR2 and CDR3 having the sequences SEQ ID nos. 7,2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 having the sequences SEQ ID nos. 9, 5 and 12, respectively; and

d) an antibody comprising heavy chain CDR1, CDR2 and CDR3 having the sequences SEQ ID nos. 8, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 having the sequences SEQ ID nos. 9, 5 and 11, respectively;

l is a linker;

d is a drug moiety of the following formula (II):

wherein:

R₂is COOH, COOCH₃Or a thiazolyl group;

R₃is H or (C)₁-C₆) An alkyl group;

R₉is H or (C)₁-C₆) An alkyl group;

m is an integer between 1 and 8;

the wavy line indicates the point of connection with L; and

n is 1 to 12.

2. The antibody-drug conjugate of claim 1, wherein Ab is selected from the group consisting of:

a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.13 and light chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 9, 5 and 11, respectively;

b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.14 and light chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 10, 5 and 11, respectively;

c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.15 and light chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 9, 5 and 12, respectively;

d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No.16 and light chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 9, 5 and 11, respectively; and

e) an antibody comprising the heavy chain variable domain of sequence SEQ ID No.17 and the light chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 9, 5 and 12, respectively.

3. The antibody-drug conjugate of claim 1, wherein Ab is selected from the group consisting of:

a) an antibody comprising the light chain variable domain of sequence SEQ ID No.18 and the heavy chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 7,2 and 3, respectively;

b) an antibody comprising the light chain variable domain of sequence SEQ ID No.19 and the heavy chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 7,2 and 3, respectively;

c) an antibody comprising the light chain variable domain of sequence SEQ ID No.20 and the heavy chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 7,2 and 3, respectively;

d) an antibody comprising the light chain variable domain of sequence SEQ ID No.21 and the heavy chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 8, 2 and 3, respectively; and

e) an antibody comprising the light chain variable domain of sequence SEQ ID No.22 and the heavy chain CDR1, CDR2 and CDR3 of sequences SEQ ID Nos. 7,2 and 3, respectively.

4. The antibody-drug conjugate of claim 1, wherein Ab is selected from I) the antibodies produced by hybridomas I-4757, I-4773, I-4775, I-4736, and I-4774 deposited at CNCM, france pasteur institute, on 30 days 5/2013, 26 days 6/2013, 24 days 4/2013, and 26 days 6/2013, respectively.

5. The antibody-drug conjugate of claim 1, wherein Ab comprises:

b) a light chain variable domain (VL) of sequence SEQ ID No.35 wherein said sequence SEQ ID No.35 comprises at least 1 back mutation selected from residues 22, 53, 55, 65, 71, 72, 77 or 87.

6. The antibody-drug conjugate of claim 1, wherein Ab is selected from the group consisting of:

a) a heavy chain variable domain comprising a sequence selected from SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence having at least 80% identity to SEQ ID nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80; and antibodies having the light chain CDR1, CDR2, and CDR3 of sequences SEQ ID nos. 9, 5, and 11, respectively;

b) a light chain variable domain comprising a sequence selected from SEQ ID No.57 or 60 or any sequence having at least 80% identity to SEQ ID No.57 or 60; and antibodies of heavy chain CDR1, CDR2 and CDR3 of sequences SEQ ID nos. 7,2 and 3, respectively; and

7. The antibody-drug conjugate of claim 1, wherein Ab comprises:

8. The antibody-drug conjugate of claim 1, wherein L is a linker of the following formula (III):

wherein

L₂Is (C)₄-C₁₀) Cycloalkyl-carbonyl group, (C)₂-C₆) Alkyl, (C)₂-C₆) An alkyl-carbonyl group, which is a carbonyl group,

w is an amino acid unit; w is an integer between 0 and 5;

y is PAB-carbonyl, wherein PAB isy is 0 or 1;

asterisks indicate points of attachment to D; and

the wavy line indicates the point of attachment to Ab.

9. The antibody-drug conjugate of claim 8, wherein L₂Is of the formula:

wherein

Star sign of AND (W)_wA point of connection; and

10. The antibody-drug conjugate of claim 8, wherein (W)_wSelected from:

a single bond, a,

Wherein

Asterisk denotes the sum (Y)_yA point of connection; and

wavy line representation and L₂The point of connection.

11. The antibody-drug conjugate of claim 8, wherein w-0; or W is 2 and (W)_wSelected from:

wherein

Asterisk denotes the sum (Y)_yA point of connection; and

wavy line representation and L₂The point of connection.

12. The antibody-drug conjugate of claim 1, wherein (L-D) is selected from:

where the wavy line indicates the point of attachment to the Ab.

13. The antibody-drug conjugate of claim 1, having a formula selected from the group consisting of:

wherein Ab is selected from the group consisting of I) the antibodies produced by hybridomas I-4757, I-4773, I-4775, I-4736 and I-4774 deposited at CNCM, Pasteur research institute, France, on 30 days 5/2013, 26 days 6/2013, 24 days 4/2013 and 26 days 6/2013, respectively.

14. The antibody-drug conjugate of claim 13, wherein Ab is selected from the group consisting of:

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.56 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.56 and a light chain variable domain of sequence SEQ ID No. 60;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.62 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.64 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.64 and a light chain variable domain of sequence SEQ ID No. 60;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.66 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.68 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.68 and a light chain variable domain of sequence SEQ ID No. 60;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.70 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.72 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.74 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.76 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.78 and a light chain variable domain of sequence SEQ ID No. 57;

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.78 and a light chain variable domain of sequence SEQ ID No. 60; and

-an antibody comprising a heavy chain variable domain of sequence SEQ ID No.80 and a light chain variable domain of sequence SEQ ID No. 57.

15. The antibody-drug conjugate of claim 14, wherein Ab is selected from the group consisting of:

-an antibody having a heavy chain of sequence SEQ ID No.58 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.58 and a light chain of sequence SEQ ID No. 61;

-an antibody having a heavy chain of sequence SEQ ID No.63 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.65 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.65 and a light chain of sequence SEQ ID No. 61;

-an antibody having a heavy chain of sequence SEQ ID No.67 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.69 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.69 and a light chain of sequence SEQ ID No. 61;

-an antibody having a heavy chain of sequence SEQ ID No.71 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.73 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.75 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.77 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.79 and a light chain of sequence SEQ ID No. 59;

-an antibody having a heavy chain of sequence SEQ ID No.79 and a light chain of sequence SEQ ID No. 61; and

an antibody having a heavy chain of sequence SEQ ID No.81 and a light chain of sequence SEQ ID No. 59.

16. The antibody-drug conjugate of claim 13, having the formula:

or a pharmaceutically acceptable salt thereof,

wherein Ab is an antibody having a heavy chain variable domain of sequence SEQ ID No.80 and a light chain variable domain of sequence SEQ ID No. 57.

17. The antibody-drug conjugate of claim 16, wherein Ab is an antibody having a heavy chain of sequence SEQ ID No.81 and a light chain of sequence SEQ ID No. 59.

18. The antibody-drug conjugate of claim 1, wherein n is 2.

19. The antibody-drug conjugate of claim 1, wherein n is 4.

20. The antibody-drug conjugate of any one of the preceding claims for use as a medicament.

21. A composition comprising at least one antibody-drug conjugate of any one of the preceding claims.

22. The composition of claim 21, further comprising a pharmaceutically acceptable carrier.

23. The composition of claim 21 for use in treating an IGF-1R-expressing cancer.

24. The composition of claim 23, wherein the IGF-1R-expressing cancer is selected from the group consisting of breast cancer, colon cancer, esophageal cancer, hepatocellular cancer, gastric cancer, glioma, lung cancer, melanoma, osteosarcoma, ovarian cancer, prostate cancer, rhabdomyosarcoma, renal cancer, thyroid cancer, endometrial cancer, mesothelioma, oral squamous cell carcinoma, and any drug-resistant cancer.

25. Use of at least one antibody-drug conjugate according to any one of claims 1 to 19 for the preparation of a medicament for the treatment of cancer expressing IGF-1R.