EA041532B1 - HUMANIZED ANTIBODIES AGAINST CLEVER-1 AND THEIR APPLICATIONS - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to agents specific for the CLEVER-1 protein by recognizing a specific CLEVER-1 epitope and their uses.

State of the art invention

The contents of publications and other materials used in the present description to illuminate the prior art for the invention, and in particular, cases of communication of additional details in relation to practice, are given by reference.

CLEVER-1 is a protein described in WO 03/057130, Common Lymphatic Endothelial and Vascular Endothelial Receptor-1. It is a binding protein that mediates the adhesion of lymphocytes to the endothelium in both the systemic and lymphatic vasculature. By blocking the interaction of CLEVER-1 and its lymphocyte substrate, lymphocyte recycling and lymphocyte migration and related conditions such as inflammation at the site of lymphocyte inflow and outflow can be simultaneously controlled. WO 03/057130 further describes that CLEVER-1 mediates the binding of other types of leukocytes, such as monocytes and granulocytes, to HEV-like vessels. Thus, by blocking the interaction of CLEVER-1 and cancer cells, it becomes possible to control metastasis by preventing lymphatics from taking up cancer cells that bind to CLEVER 1, and thus preventing cancer from spreading to the lymph nodes.

Overview of CLEVER-1, i.e. stabilin-1, see Kzhyshkowska J. (2010), The ScientificWorld JOURNAL 10, 2039-2053. Suppression of Th1 lymphocytes by CLEVER-1 has recently been described in Palani et al. (2016), Journal of Immunology 196: 115-123.

WO 2010/122217 describes a subtype of macrophages in tumors, in the placenta and in the blood of pregnant women. This macrophage subtype was identified as a CLEVER-1 positive macrophage and presumably a type 3 macrophage. counteraction or stimulation, respectively, in relation to the CLEVER-1 receptor in this cell of the immune system, you can influence the individual. Counteracting or downregulating the receptor reduces the size of the cancer and/or the growth of the cancer. Stimulation or upregulation of the receptor can be used to obtain fetomaternal tolerance and to prevent pregnancy complications.

Mechanisms for tumor-associated macrophages (TAMs) are also described in Noy R. and Pollard J. W., Tumour-Associated Macrophages: From Mechanisms to Therapy, published in Immunity 41, July 17, 2014, p. 49-61. M2 macrophages predominate in human cancers and stimulate tumor growth, however, these tumor-stimulating macrophages can be modulated into tumor growth-inhibiting macrophages, also called M1 macrophages or pro-inflammatory macrophages, to slow or stop the growth of malignant tumors. However, it has been noted that attempts to treat malignant tumors using currently available drugs designed to target TAMs have been accompanied by undesirable side effects, for example macrophage therapies may have systemic toxicity or paradoxically promote tumor growth because they target for all macrophages.

Particularly preferred CLEVER-1 antagonist monoclonal antibodies 3266 (DSM ACC2519) and 3-372 (DSM ACC2520), both deposited under the terms of the Budapest Agreement on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on August 21, 2001, at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, are described in WO 03/057130.

Purpose and essence of the invention

One of the objectives of the present invention is to provide an agent capable of binding to a specific human CLEVER-1 epitope. In particular, it has been found that an agent capable of binding to a specific human CLEVER-1 epitope can be used to activate macrophages to switch their phenotype from M2 macrophages to M1 macrophages.

Furthermore, it is an object of the invention to provide a humanized antibody or a humanized single chain Fv or Fab fragment for binding to human CLEVER-1 with increased binding activity compared to the monoclonal antibody 3-372 (DSM ACC2520 deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH August 21, 2001).

Thus, the present invention relates to an agent capable of binding to a human CLEVER-1 epitope that is not contiguous and contains the sequences:

PFTVLVPSVSSFSSR (SEQ ID NO: 1) and

QEITVTFNQFTK (SEQ ID NO: 2).

In particular, the present invention relates to an agent capable of binding to human CLEVER-1, recognizing a CLEVER-1 epitope that is not contiguous and contains the sequences:

- 1 041532

PFTVLVPSVSSFSSR (SEQ ID NO: 1), and

QEITVTFNQFTK (SEQ ID NO: 2) and the agent contains sequences of complementarity determining regions (CDRs) binding to sequences of the specified epitope selected from the group consisting of

TSGMGIG (SEQ ID NO: 7),

HIWWDDDDKRYNPALKS (SEQ ID NO: 8),

HYGYDPYYAMDY (SEQ ID NO: 9),

TASSSVSSSYLH (SEQ ID NO: 10),

RTSNLAS (SEQ ID NO: 11) and

HQYHRSPPT (SEQ ID NO: 12).

According to the invention, an agent capable of binding to human CLEVER-1 recognizing the CLEVER-1 epitope as defined herein may be selected from the group consisting of an antibody, a single chain Fv or Fab fragment(s), a peptide(s), or any another macromolecule having the appropriate affinity for binding to the specified epitope.

In one aspect, the present invention relates to an agent capable of binding to human CLEVER1 in an individual, for use in terminating tumor- or antigen-induced immunosuppression by modulating translation of M2 macrophages to M1 macrophages, wherein the agent binds to a human CLEVER-1 epitope that is not continuous and contains the sequences:

PFTVLVPSVSSFSSR (SEQ ID NO: 1) and

QEITVTFNQFTK (SEQ ID NO: 2).

An agent of the invention capable of binding to human CLEVER-1 on TAM, preferably specific epitope sequences on CLEVER-1, is useful for treating or preventing cancer by reducing the size of the cancer; by reducing the growth of a malignant tumor in an individual; and/or by inhibiting the migration of malignant cells and the formation of metastases, where the immune suppression in relation to the growth of malignant neoplasms is stopped by modulating translation of M2 macrophages into M1 macrophages.

An agent of the invention capable of binding to human CLEVER-1, preferably specific epitope sequences on CLEVER-1, is also suitable for use in the treatment of chronic infections in an individual where immune suppression against infectious antigens is halted by translation via modulation of M2 macrophages into macrophages M1.

An agent of the invention capable of binding to human CLEVER-1, preferably specific epitope sequences on CLEVER-1, is also suitable for use as a vaccine adjuvant, where suppression of immunity against vaccine antigens is halted by modulating translation of M2 macrophages to M1 macrophages.

In another aspect, the invention relates to a humanized antibody or single chain Fv or Fab fragment capable of binding to a human CLEVER-1 epitope that is not contiguous and contains the sequences:

PFTVLVPSVSSFSSR (SEQ ID NO: 1) and

QEITVTFNQFTK (SEQ ID NO: 2) and said antibody or single chain Fv or Fab fragment contains:

a) human IgG4 kappa heavy chain and light chain constant regions, and

b) one or more of the following sequences of complementarity determining regions (CDRs)

i) heavy chain

CDR 1: TSGMGIG (SEQ ID NO: 7), and/or

CDR 2: HIWWDDDKRYNPALKS (SEQ ID NO: 8), and/or

CDR 3: HYGYDPYYAMDY (SEQ ID NO: 9); and ii) light chain

CDR 1: TASSSVSSSYLH (SEQ ID NO: 10), and/or

CDR 2: RTSNLAS (SEQ ID NO: 11), and/or

CDR 3: HQYHRSPPT (SEQ ID NO: 12).

Another object of the present invention is also to provide a pharmaceutical composition comprising an agent capable of binding to human CLEVER-1 or a humanized antibody or single chain Fv or Fab fragment of the invention and a suitable excipient.

The present invention also provides a pharmaceutical composition comprising an agent capable of binding to human CLEVER-1, or a humanized antibody or single chain Fv or Fab fragment as defined above, and a suitable excipient for use in terminating tumor or antigen induced immunosuppression.

The pharmaceutical composition of the invention is suitable for use in the treatment

- 2 041532 or preventing a malignant tumor by reducing the size of a malignant tumor; by reducing the growth of a malignant tumor in an individual; and/or by inhibiting the migration of malignant cells and the formation of metastases. The pharmaceutical composition of the invention is also suitable for use in the treatment of chronic infections in an individual or for use as a vaccine adjuvant.

Brief description of the drawings

Fig. 1A and 1B illustrate a heatmap representation of the results obtained for antibody 3-266 and antibody AK FUMM 9-11.

Fig. 2 illustrates a heatmap representation of the results obtained for the FU-HI-3-372 antibody.

In FIG. 3 schematically illustrates the domain organization of the positions in CLEVER-1 of the identified binding motifs.

Fig. 4 illustrates 1% agarose gel separation of RT-PCR products for hybridoma 3-372.

Fig. 5 illustrates a Coomassie blue stained gel after SDS-PAGE purified with Protein A of chimeric IgG4 3-372.

Fig. 6 illustrates a competitive ELISA of CLEVER-1.

Fig. 7 illustrates the design of the Antitope pANT vector.

Fig. 8 illustrates a Coomassie blue stained SDS-PAGE gel of selected Protein A purified antibodies.

Fig. 9 illustrates a competitive ELISA of CLEVER-1.

In FIG. 10A shows the results of determining HLA-DR expression on CD14 positive cells, and Figure 10B shows the results for soluble TNF-alpha measured in culture medium using the TNF-alpha ELISA kit (Invitrogen).

In FIG. 11A shows TAM repolarization in E0771 syngeneic breast carcinomas following administration of an antibody that binds to CLEVER-1, and FIG. 11B shows increased secretion of TNFalpha on TAMs from syngeneic breast carcinoma E0771 after administration of an antibody that binds to CLEVER-1.

In FIG. 12 illustrates that CLEVER-1 binding to antibodies 9-11 and 3-372 mimics opposite effects on mTOR and c-Jun signaling in human peripheral blood monocytes.

Detailed description of the invention

Terms.

The term agent capable of binding to a human CLEVER-1 epitope refers to agents, including antibodies and fragments, peptides or the like, that are capable of binding to the specific epitope sequences defined in the present invention. The agent may also be any macromolecule having the appropriate affinity for binding to the specified epitope.

The term antibody or fragment thereof is used in its broadest sense to encompass an antibody or fragment thereof capable of binding to a CLEVER-1 molecule in an individual. In particular, it should be understood to include chimeric, humanized or primatized antibodies, as well as antibody fragments and single chain antibodies (eg Fab, Fv), provided they have the desired biological activities.

The term humanized antibody refers to any antibody in which the constant regions of non-human antibodies are completely replaced with the human form of constant regions, and at least a portion of the variable regions from non-human antibodies, excluding three loops from the amino acid sequences outside of each variable region, that bind to the target structure are completely or partially replaced by the corresponding parts of human antibodies. Thus, in particular, any antibody named according to the naming scheme of World Health Organization International Nonproprietary Names (INN) or drug names officially assigned by the appropriate United States organization (USAN), with parts of the stem -xisu- or -zu-, in this invention is designated as a humanized antibody.

The term variable domain, also referred to as the Fv region, is the most important region for antigen binding. To provide specificity, β-chain variable loops, three on each light (VL) and heavy (V _H ) chain, are responsible for antigen binding. These loops are referred to as complementarity determining regions (CDRs).

The term single chain Fv fragment or scFv refers to fragments obtained by connecting the VH and VL domains via a linker into one polypeptide. The term humanized single chain Fv or scFv fragment refers, by analogy to the definition of the term humanized antibody above, to any single chain Fv or scFv fragment in which the constant regions originating from non-human antibodies are completely replaced by the human form of the constant regions, and at least portions of variable regions originating from unrelated

- 3 041532 human antibodies, excluding three loops from the amino acid sequences outside of each variable region that bind to the target structure, are completely or partially replaced by the corresponding parts of human antibodies.

The term Fab fragment refers to the region of an antibody that binds to antigens. The term humanized Fab fragment refers, also by analogy with the definition of the term humanized antibody above, to any Fab fragment in which constant regions derived from non-human antibodies are completely replaced by human form constant regions, and at least a portion of the variable regions derived from non-human antibodies, excluding three loops of amino acid sequences outside of each variable region that bind to the target structure, are completely or partially replaced by the corresponding parts of human antibodies.

The term peptide refers to any peptide containing one or more amino acid sequences of complementarity determining regions (CDRs) as defined herein and capable of binding to at least one human CLEVER-1 epitope.

Preferred Embodiments

One embodiment of the present invention relates to an agent capable of binding to human CLEVER-1 recognizing a CLEVER-1 epitope that is not contiguous and contains the amino acid sequences

PFTVLVPSVSSFSSR (SEQ ID NO: 1) and

QEITVTFNQFTK (SEQ ID NO: 2) from human CLEVER-1, and said agent contains one or more amino acid sequences of complementarity determining regions (CDRs) that bind to said epitope sequences selected from the group consisting of

TSGMGIG (SEQ ID NO: 7),

HIWWDDDDKRYNPALKS (SEQ ID NO: 8),

HYGYDPYYAMDY (SEQ ID NO: 9),

TASSSVSSSYLH (SEQ ID NO: 10),

RTSNLAS (SEQ ID NO: 11) and

HQYHRSPPT (SEQ ID NO: 12).

In some preferred embodiments of the present invention, the non-contiguous human CLEVER-1 epitope further comprises one or more amino acid sequences selected from the group consisting of

ATQTGRVFLQ (SEQ ID NO: 3),

DSLRDGRLIYLF (SEQ ID NO: 4),

SKGRILTMANQVL (SEQ ID NO: 5) and

LCVYQKPGQAFCTCR (SEQ ID NO: 6).

Part of the human CLEVER-1 target protein, i. human stabilin-1, as defined in SEQ ID NO: 31. Epitopes from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 on CLEVER-1 correspond to amino acids 420-434, 473-484, 390-399, 576-587, 615-627 and 313-327 from the human CLEVER-1 target protein as defined in SEQ ID NO: 31.

In some preferred embodiments of the present invention, an agent capable of binding to a human CLEVER-1 epitope comprises at least two, preferably three, more preferably four, even more preferably five, and most preferably all six amino acid sequences of complementarity determining regions (CDRs) defined by higher.

In accordance with the present invention, the agent capable of binding to human CLEVER-1 may be selected from the group consisting of an antibody, single chain Fv or Fab fragment(s), peptide(s) or macromolecule(s).

In some preferred embodiments of the present invention, the agent capable of binding to human CLEVER-1 is a humanized antibody or single chain Fv or Fab fragment, and said antibody or humanized single chain Fv or Fab fragment contains

a) human IgG kappa heavy chain and light chain constant regions, and

b) one or more of the following sequences of complementarity determining regions (CDRs)

i) heavy chain

CDR 1: TSGMGIG (SEQ ID NO: 7), and/or

CDR 2: HIWWDDDKRYNPALKS (SEQ ID NO: 8), and/or

CDR 3: HYGYDPYYAMDY (SEQ ID NO: 9); and ii) light chain

CDR 1: TASSSVSSSYLH (SEQ ID NO: 10), and/or

CDR 2: RTSNLAS (SEQ ID NO: 11), and/or

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CDR 3: HQYHRSPPT (SEQ ID NO: 12).

A humanized antibody or single chain Fv or Fab fragment capable of binding to a human CLEVER-1 epitope recognizes sequences of a non-contiguous epitope as defined above. The human CLEVER-1 non-contiguous epitope comprises at least the sequences of SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the human CLEVER-1 non-contiguous epitope further comprises one or more sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

In some of the above embodiments of the present invention, at least two, preferably three, more preferably four, even more preferably five, and most preferably all six CDRs as defined above are contained in a humanized antibody or single chain Fv or Fab fragment. .

In some embodiments, the human IgG heavy chain variable region sequence from the humanized antibody or single chain Fv or Fab fragment is selected from the group consisting of SeQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO : 20, preferably SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20. In some embodiments of the present invention, the human IgG light chain variable region sequence from a humanized antibody or single chain Fv or Fab fragment is selected from the group, consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30, preferably SEQ ID NO: 30.

In many embodiments of a humanized antibody or single chain Fv or Fab fragment of the invention, the human IgG kappa heavy chain and light chain constant regions are as follows. Human IgG4 constant regions are preferred. Many preferred embodiments contain human IgG4 heavy chain and IgG4 kappa light chain with L248E and/or preferably S241P mutations.

In some embodiments, the humanized antibody or single chain Fv or Fab fragment is capable of binding to human CLEVER-1 with a relative IC50 < 1.0, preferably < 0.8, more preferably < 0.6, and most preferably < 0.5 compared to the IC50 of monoclonal antibody 3-372 (DSM ACC2520, deposited with DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH August 21, 2001).

In accordance with one embodiment of the invention, the combination of heavy and light chain human IgG variable regions is selected from the combinations shown in Table 1. 5 capable of binding to human CLEVER-1 with a relative IC50<1.0 compared to the IC50 of monoclonal antibody 3-372 (DSM ACC2520, deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH August 21, 2001).

The pharmaceutical composition of the invention contains an agent capable of binding to human CLEVER-1 or a humanized antibody or single chain Fv or Fab fragment as described above and a suitable excipient.

Transition via modulation of tumor-stimulating macrophages (M2) to pro-inflammatory macrophages (M1).

It has also been found that an agent capable of binding to human CLEVER-1, especially specific epitope sequences on CLEVER-1 defined in the present invention, can be used to activate macrophages to switch their phenotype from M2 macrophages to M1 macrophages. In particular, an agent capable of binding to CLEVER-1 on the TAM can be used to effect a modulation translation of tumor-stimulating macrophages (M2) to pro-inflammatory macrophages (M1). This modulation increases the activation of T cells and ultimately leads, for example, to the cessation of cancer-derived immunosuppression. More specifically, it has been found that an agent capable of binding to specific sequences on the CLEVER-1 molecule can be used to terminate immunosuppression by modulating M2 macrophages to M1 macrophages. Therefore, the present invention provides a method for influencing the immune system of an individual, and is particularly useful for treating cancer or preventing metastasis, but is not limited to this method.

Macrophages can be divided into two distinct phenotypes: M1 and M2 macrophages. M1 macrophages are classic pro-inflammatory macrophages that produce large amounts of pro-inflammatory cytokines and co-stimulatory molecules and are very effective in activating T cell responses. In contrast, M2 macrophages are immunosuppressive cells that synthesize anti-inflammatory cytokines and induce regulatory T cells and thus greatly attenuate antigen-driven T cell activation. Tumor-associated macrophages (TAMs) are considered dangerous because they mature into M2 macrophages (tumor-promoting macrophages) in the tumor environment and suppress the immune response against tumors and mediate angiogenic switching, a critical step in cancer growth. M2 macrophages can be translated through modulation into M1 macrophages (pro-inflammatory macrophages), and this

- 5 041532 rotation of the phenotype from M2 to M1 can directly or indirectly cause tumor rejection.

In the present context, the expression M1 macrophages or pro-inflammatory macrophages refers to macrophages characterized by an increased measured level of macrophage/monocyte TNF-alpha (TNF-α) secretion or HLA-DR expression. Modulating conversion of M2 macrophages to M1 macrophages may increase monocyte TNF-alpha secretion as well as HLA-DR expression compared to control values measured prior to administration of an agent capable of binding to human CLEVER-1, or to values from one or more previous measurements conducted at different time points in the same patient. It is important to compare the measured values of monocyte TNF-alpha secretion and HLA-DR expression with values for the same patient, since the level of these markers may vary from individual to individual, and for example, cytokines such as interferon-gamma and activation by LPS may increase expression of TNF-alpha by M2 macrophages.

Surprisingly, it has been found that M2 macrophages can be activated to modulate into M1 macrophages by contacting said macrophages with an agent capable of binding to human CLEVER-1, such as an antibody or fragment thereof, peptide(s) or macromolecule(s), as defined in the present invention. In particular, it has been found that cancer-associated M2 macrophages can be modulated or repolarized into M1 macrophages by contacting said macrophages with an agent capable of binding to human CLEVER-1 on the TAM. Both phenotypes can be present at the same time, and both phenotypes can be found in tumors.

An agent capable of binding to human CLEVER-1, such as an antigen or fragment thereof, peptide(s), or macromolecule(s), binds to human CLEVER-1 to achieve such modulation or repolarization of macrophage phenotypes. Agents specific for the CLEVER-1 protein have been identified to recognize the specific CLEVER-1 epitope sequences defined in the present invention.

Specific binding to two or more of these epitope sequences on CLEVER-1 on TAM may provide a new way to treat cancer or prevent metastasis without dangerous side effects, since the treatment can be targeted to specific epitopes to achieve the desired modulation of the macrophage phenotype. Therefore, the discoveries described herein are particularly useful for the treatment or prevention of all cancers associated with increased numbers of tumor-promoting macrophages or other pathologies such as chronic inflammation when the individual is immunosuppressed-dominated. Therefore, a method for treating cancer or preventing metastasis comprises administering to an individual an agent capable of binding to human CLEVER-1, preferably specific epitopes on the CLEVER-1 molecule as defined above. The method includes treating or preventing a cancer by reducing the size of the cancer and/or; by reducing the growth of a malignant tumor in an individual; and/or by inhibiting the migration of malignant cells and the formation of metastases. Thus, a benign or malignant tumor or metastasis of a malignant tumor such as skin cancer and colon cancer can be treated. Leukemias, lymphomas and multiple myelomas can also be treated. In particular, melanomas and lymphomas are expected to respond very well to treatment based on animal models.

Macrophages also play an important role in the resolution of inflammation and infection, in addition to influencing the growth or regression of tumors. During infections, a switch from M1 to M2 macrophages can occur, leading to the formation of a suppressive environment that suppresses effector immunity. Therefore, the findings described herein for modulating macrophage phenotype can be used in the treatment of chronic infections to reverse immune suppression against infectious antigens. The invention also relates to a method for the treatment of chronic infections comprising administering to an individual an agent capable of binding to CLEVER-1, preferably two or more specific epitope sequences on the CLEVER-1 molecule defined in the present invention, wherein said agent can activate macrophages to switch them phenotype from M2 to M1.

In addition, an agent capable of binding to the CLEVER-1 molecule on macrophages and monocytes in an individual can be used as an adjuvant in vaccines. Said agent provides repolarization of the macrophages and thus terminates or at least reduces the suppression of immunity against vaccine antigens. Any antigen-induced vaccination may provide an advantage if immunosuppressive elements can be temporarily removed from the host or vaccination site.

Translation by macrophage modulation of M2 to M1 can be verified by measuring the secretion of monocyte TNF-alpha from human blood samples. Therefore, increased secretion of TNF-alpha can be used as a marker to monitor an individual's response to treatment. The secretion of TNF-alpha can be determined in peripheral blood monocytes enriched from blood

- 6 041532 vi selected from the patient. The measured level of TNF-alpha can be used as a marker for patient response to treatment involving the administration of an agent capable of binding to CLEVER-1 in the patient, where the level is compared with a control level measured in the patient prior to administration of said agent to the patient, or with values of one or several previous measurements taken at different time points in the same patient.

Method for determining the effectiveness of therapy against CLEVER-1 by monitoring the development of the transition through the modulation of M2 macrophages to M1 macrophages, when an agent capable of binding to CLEVER-1, preferably with the specified one, two or more specific epitope sequences on CLEVER-1, is administered in a patient , including the stages:

(a) obtaining peripheral blood monocytes (PBLs) from a blood sample collected from said patient, (b) measuring TNF-α secretion from said PBLs, and/or (c) measuring HLA-DR expression on CD14 positive PBLs, and (e ) comparing the levels of TNF-α secretion and/or HLA-DR expression measured in steps (b) and (c) with control values to determine the effectiveness of treatment against CLEVER-1, where control values are values measured before administration of the agent, capable of binding to CLEVER-1 in a patient, or the value of one or more previous measurements taken at different time points in the same patient, and where increased TNF-alpha secretion or HLA-DR expression is indicative of conversion via modulation of M2 macrophages to macrophages M1.

Determination of TNF-alpha secretion from peripheral blood monocytes obtained from a blood sample taken from a patient can be carried out by conventional methods, for example using a commercial TNF-alpha ELISA kit. HLA-DR expression on CD14 positive monocytes can also be monitored using the known flow cytometry method.

The progression of translation by modulating M2 macrophages to M1 macrophages can be monitored by comparing the level of monocyte TNF-alpha secretion with control values measured prior to administration of the CLEVER-1 binding agent in the patient, or with the values of one or more previous measurements taken in others. time points in the same patient. For example, a decrease in monocyte TNF-alpha secretion compared to prior measurement or control can be used as an indicator of higher M2 macrophage protein expression, while an increase in TNF-alpha compared to prior measurement or control can be used. be used as an indicator of higher expression of M1 macrophage proteins with lower expression of M2 macrophage proteins, where it can also be used as an indicator of the effectiveness of treatment against CLEVER-1. An increased level of TNF-alpha indicates higher expression of M1 macrophage proteins with lower expression of M2 macrophage proteins, i.e. this can be attributed to the ability to respond to said therapy. An agent capable of binding to CLEVER-1 can activate at least a portion of M2 macrophages to repolarize into M1 macrophages, and both macrophage phenotypes may be present after administration of said agent, but an increase in expression of M1 macrophage proteins can be observed compared to the situation before administration of said agent . Typically, at least a two-fold increase in measured TNF-alpha secretion compared to the control value is indicative of modulation of M2 macrophages to M1 macrophages and thus is indicative of the patient's ability to respond to therapy.

Diseases responding to treatment.

The balancing of immune activation and suppression is very critical for human (or animal) homeostasis in the fight against foreign material formed in the human (or animal) body or entered into the human (or animal) body. An example from Palani et al. (2016) provides a physiological example of this and shows that local immunosuppression is critical for the well-being of the embryo in an environment with dominant maternal immune defenses. The same may be the case in chronic infections, as some pathogens (such as in tuberculosis) have learned to use a similar mechanism to hide from the host's immune system and can form sites of chronic infection (in hepatitis). Removal of this local immunosuppression may help the host fight these infections, as well as improve vaccination against these resistant pathogens.

Tumors have also adapted this immunosuppression to their advantage. The method according to the present invention for treating or preventing a cancer by reducing the size of a cancer; by reducing the growth of a malignant tumor; and/or by inhibiting the migration of malignant cells and the formation of metastases is applicable to all forms of malignant tumors. Thus, a benign or malignant tumor or metastasis of a malignant tumor such as skin cancer and colon cancer can be treated. Leukemias, lymphomas, and multiple myelomas can also be treated. IN

- 7 041532 in particular, it is expected that melanomas and lymphomas may respond very well to treatment based on animal models.

The present inventors believe that an agent capable of binding to CLEVER-1, or a humanized antibody, or a single chain Fv, or a Fab fragment according to the present invention, or a pharmaceutical composition according to the present invention are useful in the treatment or prevention of all types of sarcomas, e.g. fibrosarcomas, liposarcomas, chondrosarcomas, osteosarcomas, angiosarcomas, lymphangiosarcomas, leiomyosarcomas and rhabdomyosarcomas, mesotheliomas, meningiomas, leukemias, lymphomas, as well as all types of carcinomas such as squamous cell carcinoma, basal cell carcinoma, adenocarcinomas, papillary carcinomas, cystadenocarcinomas, bronchogenic carcinomas, melanomas, types of renal cell carcinomas, hepatic cell carcinomas, transitional cell carcinomas, choriocarcinomas, seminomas, and embryonic carcinomas.

An agent capable of binding to human CLEVER-1 in an individual, or a humanized antibody, or a single chain Fv, or a Fab fragment, or a pharmaceutical composition of the invention are useful for terminating tumor- or antigen-induced immunosuppression by modulating translation of M2 macrophages to M1 macrophages where the agent binds to the human CLEVER-1 epitope sequences defined in the present invention.

An agent capable of binding to human CLEVER-1 in an individual, or a humanized antibody, or a single chain Fv, or a Fab fragment, or a pharmaceutical composition of the invention are useful for treating or preventing cancer by downsizing the cancer; by reducing the growth of a malignant tumor in an individual; and/or by inhibiting the migration of malignant cells and the formation of metastases, where immunosuppression in the area of growth of the malignant tumor is stopped by modulating translation of M2 macrophages to M1 macrophages.

An agent capable of binding to human CLEVER-1 in an individual, or a humanized antibody, or a single chain Fv, or a Fab fragment, or a pharmaceutical composition of the invention are also suitable for use in the treatment of a chronic infection in an individual, where suppression of immunity against infectious antigens is stopped by translation by modulation of M2 macrophages into M1 macrophages.

An agent capable of binding to human CLEVER-1 in an individual, or a humanized antibody, or a single chain Fv, or a Fab fragment, or a pharmaceutical composition of the invention are also suitable for use as a vaccine adjuvant, where suppression of immunity against vaccine antigens is terminated by modulation-assisted translation macrophages M2 to macrophages M1.

The modulation method for converting M2 macrophages to M1 macrophages comprises administering to a subject in need thereof an agent capable of binding to CLEVER-1, preferably capable of binding to specific sequences on the CLEVER-1 molecule as defined in the present invention. This method can be used in the treatment of a malignant tumor or in the prevention of metastasis in an individual, or in the treatment of chronic infections in an individual. Response to treatment can be verified by measuring TNF-α secretion from said PBLs and/or HLA-DR expression on CD14 positive PBLs as described herein.

Routes of administration, formulations and required dose

Pharmaceutical compositions for use in the present invention may be administered by any means to achieve their intended purpose. For example, the administration may be intravenous, intra-articular, intratumoral, or subcutaneous. In addition to the pharmacologically active compounds, the pharmaceutical preparations of the compounds preferably contain suitable pharmaceutically acceptable carriers containing excipients and adjuvants which facilitate the processing of the active compounds into formulations that can be used in pharmaceuticals.

The selected dose should be therapeutically effective for the disease being treated.

Accordingly, immunosuppression should be sufficient to treat the disease without effects that pose a significant threat to the desired outcome of treatment. During the treatment or prevention of cancer, the dose should be sufficient to reduce the size of the cancer, reduce the growth of the cancer, and/or inhibit cancer cell migration and metastasis formation. The dose is dependent on the turnover of the administered agent, as a rule, these treatments follow a regimen of 1-5 mg/kg every 2-4 weeks.

- 8 041532

Examples

The following experimental section illustrates the invention by providing examples.

Examples 1-9 illustrate the mapping of a non-contiguous human CLEVER-1 epitope and the production of humanized antibodies from the mouse monoclonal antibody 3-372 (DSM ACC2520, deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 21, 2001) using composite technology. Composite Human Antibody™. The ability of antibodies against CLEVER-1 to stimulate immune activation is illustrated in examples 10-13.

Example 1 illustrates a complete non-contiguous epitope mapping for antibodies targeting human CLEVER-1.

Examples 2-5 illustrate the sequencing of the heavy and light chain V region (VH and VK) of anti-Clever 1 clone 3-372 and the production of chimeric antibodies containing the 3-372 variable regions and human IgG4 kappa heavy chain and light chain constant regions. mRNA was isolated from clone hybridoma 3-372, subjected to reverse transcription, amplified by PCR and cloned specific for the antibody transcripts. The nucleotide and amino acid sequences of the heavy and light chain variable regions of the antibody were determined, and sequence data were analyzed for humanization using Antitope's proprietary Composite Human Antibody™ antibody technology.

Examples 2-5 show that the variable regions from the mouse anti-Clever 1 antibody 3-372 were cloned and sequenced. The variable region genes were combined with human IgG4(S241P) kappa heavy chain and light chain constant regions and expressed in NS0 cells to generate a chimeric anti-Clever 1 antibody. Clever-1 is similar in efficacy to the original mouse antibody.

Examples 6-9 illustrate the design of Composite Human Antibodies™ against CLEVER-1, which was expressed and tested for binding to human Clever-1. Key residues involved in the structure and binding of anti-CLEVER-1 antibodies were determined by structure and homology modeling to obtain a residue restriction map. The residue restriction map was used as a template for human region V sequence source fragments from databases containing unrelated human antibody sequences. Each selected sequence fragment, as well as junctions between fragments, was tested for the presence of potential T-cell epitopes using in silico analysis (iTope™ and TCED™). Using this method, all sequence variants of the Composite Human Antibody™ were designed to avoid T-cell epitopes. The genes for the V regions of the Composite Human Antibody™ were generated using synthetic oligonucleotides encoding combinations of selected human sequence fragments. They were then cloned into vectors containing human IgG4(S241P) kappa heavy chain and light chain constant regions, and antibodies were generated and tested for binding to the target antigen by competitive ELISA against the original reference mouse monoclonal antibody.

Examples 6-9 show the construction of the V regions of four VH and five Vk composite human antibodies Composite Human Antibody™. Combinations of contiguous heavy and light chains were expressed in NS0 cells, purified and tested for binding to CLEVER-1 in a competitive ELISA. The results indicated that the binding performance of many of the Composite Human Antibodies™ to CLEVER-1 was at least as good as that of the chimeric reference antibody, and several were markedly better. Based on the absence of a potential glycosylation site and unpaired cysteine, and the resulting expression and binding efficiency datasets, the three potential lead candidates are designated as follows: VH2/VK5, VH3/VK5, and VH4/VK5.

Examples 10-11 illustrate the binding of anti-CLEVER-1 antibody to human peripheral blood monocytes and the activation of TNF-alpha secretion by human peripheral blood monocytes.

Example 12 illustrates the mechanism of action of anti-CLEVER-1 antibodies on tumor-associated macrophages in murine models of syngeneic malignancies.

Example 13 illustrates that binding of CLEVER-1 to antibodies 9-11 and 3-372 stimulates opposite effects on mTOR and c-Jun signaling in human peripheral blood monocytes.

Example 1

Complete mapping of the non-contiguous human CLEVER-1 epitope.

Preliminary non-contiguous epitopes for four antibodies were determined using the Pepscan assay. Antibodies FAR02 VH3/VK5 and FU-HI-3-372 target the same

- 9 041532 being a continuous epitope, while antibodies 3-266 and AK FUMM 9-11 target other separate epitopes.

The study was carried out at Pepscan Presto BV (Zuidersluisweg 2, 8243RC Lelystad, The Netherlands).

Antibodies 3-266, FAR02 VH3/VK5, FU-HI-3-372 and AK FUMM 9-11 were provided by Faron Pharmaceuticals Oy.

Human CLEVER-1 target protein, i.e. human stabilin-1, as defined in SEQ ID NO: 31. Disulfide bridges link residues (Uniprot STAB1 HUMAN numbering):

112 126 I 120 136 | 138 147 | 160 171 | 164 181 | 183192

199 210 I 204 217 | 236 247 | 241 257 | 259 270 | 732746

740 756 I 758 767 | 822 837 | 831 846 | 865 879 | 873889

891 902 I 908 922 | 916 932 | 934 945 | 951 964 | 958974

CLIPS technology.

CLIPS technology uses structurally fixed peptides in defined three-dimensional structures. This results in functional mimetics of even the most complex binding sites. The CLIPS technology is currently in common use for shaping peptide libraries into single, double or triple loop structures, as well as sheet-like and helix-like folds. The CLIPS reaction occurs between the bromine groups of the CLIPS backbone and the thiol side chains of cysteine residues. The reaction is fast and specific under mild conditions. Using this simple chemical reaction, native protein sequences are transformed into CLIPS constructs with a range of structures. (Timmerman et al., J. Mol. Recognit. 2007; 20: 28329)

CLIPS library screening begins by converting the target protein into a library of up to 10,000 overlapping peptide constructs using a combinatorial array design. A matrix of linear peptides is synthesized on a solid support and then shaped into spatially defined CLIPS constructs. Constructs representing both parts of a non-contiguous epitope in the correct conformation bind the antibody with high affinity, which is detected and quantified. Constructs presenting an incomplete epitope bind the antibody with lower affinity, while constructs lacking an epitope do not bind at all.

Affinity information is used in round robin screenings to determine in detail the sequences and conformations of epitopes. A target protein containing a non-contiguous conformational epitope is transferred to a template library. Combinatorial peptides are synthesized on a proprietary minimap and chemically translated into spatially defined CLIPS constructs.

Heat map analysis.

A heat map is a graphical representation of data, where values obtained through variables are represented as colors on a two-dimensional map.

For CLIPS peptides with two loops, such a two-dimensional map can be derived from the independent sequences of the first and second loops. For example, the sequences of the 16 CLIPS peptides are actually a shuffling of, for example, 4 unique subsequences, for example, in loop 1, and for example, 4 unique subsequences, for example, in loop 2. Thus, the observed ELISA data can be plotted in a 4x4 matrix, where each X coordinate corresponds to the sequence of the first loop, and each Y coordinate corresponds to the sequence of the second loop.

To further facilitate visualization, the values in ELISA can be replaced with a color form from a continuous gradient. For example, unusually low values can be colored green, abnormally high values colored red, and average values colored black.

Synthesis of peptides.

To reconstruct the epitopes of the target molecule, a peptide library was synthesized. An amino-functionalized polypropylene support was prepared by grafting a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonylhexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with Nhydroxybenzotriazole (HOBt), followed by Boc cleavage using trifluoroacetic acid (TFA).

Standard Fmoc peptide synthesis was used to synthesize peptides on an amino-functionalized solid support via custom-modified JANUS fluid transfer stations (Perkin Elmer).

Synthesis of structural mimetics was performed using Pepscan's patented Chemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows peptides to be shaped into single loops, double loops, triple loops, leaf-like folds, helix-like folds, and combinations thereof. Attached to CLIPS matrices

- 10 041532 cysteine residues. The side chains of the numerous cysteine residues in the peptides are attached to one or two CLIPS templates. For example, a 0.5 mM solution of P2 CLIPS (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8)/acetonitrile (1:3 (v/v)). This solution is added to the arrays of peptides. The CLIPS template can bind to the side chains of two cysteine residues that are present in the solid phase bound peptides from the peptide arrays (455-well plate with 3 μl of wells). Peptide arrays are gently shaken in the solution for 30-60 min, completely covered with the solution. Finally, the peptide arrays are washed extensively with excess H ₂ O and sonicated in disruption buffer containing 1% SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70°C for 30 min, followed by sonication sonicated in H ₂ O for the next 45 min. Bearing T3 CLIPS peptides were prepared in a similar manner, but now with three cysteine residues.

Screening by ELISA.

The binding of the antibody to each of the synthesized peptides was tested in a PEPSCAN-based ELISA. Peptide arrays were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of the appropriate peroxidase antibody conjugate (SBA; Table 1) for one hour at 25°C. After washing, peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl/ml 3% H ₂ O ₂ were added.

After one hour, the development of staining was measured. Color development was quantified using a charge coupled device (CCD) camera and image processing system.

Table 1

Details of antibodies

Name Provider Cat. no Goat anti-human HRP conjugate Southern Biotech 2010-05 Rabbit anti-mouse IgG(J+L) HRP conjugate Southern Biotech 6175-05 Goat anti-rat IgM+IgG(H+L) HRP conjugate Southern Biotech 3010-05

Langedijk et al. (2011). Helical peptide arrays for lead identification and interaction site mapping, Analytical Biochemistry 417: 149-155.

Peptide design.

Various groups of peptides were synthesized according to the following design. It should be noted that in some groups the peptides were synthesized in a random order. The actual order of the peptides is shown below. Group 1

Mimetic type linear

Label LIN

Description Linear 15-mer peptides deduced from the human Clever-Ι target sequence with a one-residual shift.

Sequences (first 10)

QVLFKGCDVKTTFVT VLFKGCDVKTTFVTH LFKGCDVKTTFVTHV FKGCDVKTTFVTHVP KGCDVKTTFVTHVPC GCDVKTTFVTHVPCT CDVKTTFVTHVPCTS DVKTTFVTHVPCTSC

- 11 041532

Group 2

mimetic type

Label

Description

Sequences

Group 3

mimetic type

Label

Description

Sequences

VKTTFVTHVPCTSCA KTTFVTHVPCTSCAA linear

LIN.AA

Group 1 peptides, but with residues at positions 10 and 11 replaced by Ala. If native Ala occurs in any position, it is replaced by Gly. (first 10)

GAETPCNGHAACLDG LTMANQVLAAAISEE ILLPPTILPAAPKHC DRNGTCVCQAAFRGS PGYTQQGSEAAAPNP PIDPCRAGNAACHGL HTDALCSYVAAGQSR KGCDVKTTFAAHVPC CQALNTSTCAANSVK RAVGGGQRVAACPPG linear LIN20.C

Linear 20-length peptides deduced from the human Clever-Ι target sequence with a one residue shift. The residues of Cuz are protected with acetimidomethyl (Ast, denoted by "2"). (first 10) 2H2PENYHGDGMV2LPKDP2 SGWLRELPDQITQD2RYEVQ LAQH2HLHAR2VSQEGVAR2 IKKQT2PSGWLRELPDQITQ

- 12 041532

Group 4

mimetic type

Label

Description

Sequences

Group 5

mimetic type

Label

Description

linear

LIN25.C

Linear peptides of length 25 deduced from the human Clever-Ι target sequence with a one residue shift. The remains of Susa are protected by Ast ("2"). (first 10)

KKG2ASY2NQTIMEQG22KGFFGPD PD2QSV2S2VHGV2NHGPRGDGS2L GPGQSR2T2KLGFAGDGYQ2SPIDP IFPKE2VYIHDPTGLNVLKKG2ASY PTILPILPKH2SEEQHKIVAGS2VD ENFRGSA2QE2QDPNRFGPD2QSV2 QNTQ2SAEAPS2R2LPGYTQQGSE2 GRV2VAIDE2ELDMRGG2HTDAL2S APSGTPGDPKRTIGQILASTEAFSR DGMV2LPKDP2TDNLGG2PSNSTL2

Restriction peptides, mP2 CLIPS LOOP

The peptides are 17 long. Positions 2-16 are 15-mer sequences derived from the target protein. Positions 1 and 17 contain the remnants of Cuz connected by mP2 CLIPS. The native residues of Susa are protected by Ast ("2").

- 13 041532

Sequences

Group 6

mimetic type

Label

Description

Sequences

Group 7

Mimetic type (top 10)

CL2SYVGPGQSR2T2KC C2SYVGPGQSR2T2KLC CSYVGPGQSR2T2KLGC CYVGPGQSR2T2KLGFC

Linear disulfide mimetics CYS22

Linear 22-length disulfide mimetics developed from Uniprot information on disulfide bridges for human CLEVER-1. Cuz residues within the mimetic that do not participate in the formation of disulfide bridges are protected by Ast ("2"). (first 10)

WGSR2HECPGGAETP2NGHGTC SR2HECPGGAETP2NGHGTCLD 2HECPGGAETP2NGHGTCLDGM ECPGGAETP2NGHGTCLDGMDR GAETPCNGHGT2LDGMDRNGTC ETPCNGHGT2LDGMDRNGTCV2 PCNGHGT 2 LDGMDRNGTCV2 QE LDGMDRNGT2VCQENFRGSACT2QEVFRNGT2VCQENG

Combinatorial mimetics with

- 14 041532 disulfide bridges

CYS27 label

Description Combinatorial peptides of length 27. At positions 1-11 and 16-27 are 11-mer sequences derived from the target sequence on page 7 linked by a "GGSGG" linker. This group of peptides was designed based on information on disulfide bridges obtained from Uniprot. Cuz residues within the mimetic that do not participate in the formation of disulfide bridges are protected by Ast ("2").

Sequences (first 10)

PGYWGSR2HECGGSGGAETP2NGHGTC YWGSR2HECPGGGSGGAETP2NGHGTC GSR2HECPGGAGGSGGAETP2NGHGTC R2HECPGGAETGGSGGAETP2NGHGTC HECPGGAETP2GGSGGAETP2NGHGTC CPGGAETP2NGGGSGGAETP2NGHGTC PGYWGSR2HECGGSGGTP2NGHGTCLD YWGSR2HECPGGGSGGTP2NGHGTCLD GSR2HECPGGAGGSGGTP2NGHGTCLD R2HECPGGAETGGSGGTP2NGHGTCLD Группа 8

Mimetic type Non-continuous matrix,

TK CLIPS Label MAT

Description Peptides are 33 long. Positions 2-16 and 18-32 are 15-mer peptides derived from the human Clever-Ι target sequence. In positions 1, 17 and 33 are the remnants of Suz, connected by means of TK CLIPS. The native residues of Susa are protected by Ast ("2"). Sequences (first 10)

CPNRFGPD2QSV2S2VCV2S2VHGV2NHGPRGC CHGDGMV2LPKDP2TDCSAG2FAF2SPFS2DRC C2VD2QALNTST2PPNCPKH2SEEQHKIVAGSC CPKH2SEEQHKIVAGSCGPD2TQ2PGGFSNP2C CRYEVQLGGSMVSMSGCVP2TS2AAIKKQT2PC CHKIVAGS2VD2QALNCIHMLDGILLPPTILPC CF2T2RPGLVSINSNACVTADGKTS2V2RESEC C2VYIHDPTGLNVLKKCGSGGV2QQGT2APGFC CLRVAVAMMDQG2REICDGRA2YGHLLHEVQKC CYSYKYKDQPQQTFNICHEVQKATQTGRVFLQC Детали скрининга.

The binding of the antibody depends on a combination of factors including the concentration of the antibody and the amount and nature of the competing proteins in the ELISA buffer. Also, pre-coating conditions (specific processing of peptide arrays prior to incubation with an experimental sample) affect binding. These details are summarized in Table. 2. For Pepscan buffer and pre-treatment (SQ), the numbers indicate the relative amount of competing protein (combinations of horse serum and ovalbumin).

- 15 041532

table 2

Screening Conditions

Label Breeding Buffer for samples Preliminary processing 3-266 5 µg/ml 10%SQ 10%SQ AK FUMM 9-11 1 µg/ml 50%SQ 50%SQ FARO2 VH3/VK5 3 µg/ml 1%SQ 1%SQ FU-HI_3-372 5 µg/ml 1%SQ 1%SQ

Results.

Antibody 3-266.

When tested under high stringency conditions, antibody 3-266 did not bind any of the peptides present in the arrays. When tested under low stringency conditions, the antibody bound all peptides from all groups. The results obtained using simple epitope mimetics suggest that _{the 1030} QWLKSAGITLPADRR ₁₀₄₄ sequence represents the dominant portion of the epitope. Data obtained using combinatorial epitope mimetics (FIG. 1) suggest that the antibody also recognizes _{the 857} LHARCVSQEGVARCR ₈₇₁ sequence. Moreover, a weak and stable signal was recorded for peptides with the sequence ₄₃₅ TMNASLAQQLCRQHI ₄₅₀ . Fig. 1A illustrates a heatmap representation of the results obtained for antibody 3-266 in group 8 (non-contiguous epitope mimetics). The medium signal is plotted in black and the extremely high signal is plotted in light. The boxed areas are enlarged.

Antibody AK FUMM 9-11.

When tested under high stringency conditions, the AK-FUMM 9-11 antibody bound peptides from all groups. The results obtained using simple epitope mimetics suggest that the antibody recognizes ₈₈₅ PSNPCSHPDRGG ₈₉₆ representing the dominant portion of the epitope. Data obtained using combinatorial epitope mimetics suggest that the antibody also recognizes the ₁₆₆ FRGSACQECQDPNRF ₁₈₀ sequence (FIG. 1B). Fig. 1B illustrates a heat map view of the results obtained for the AK FUMM 911 antibody in group 8 (non-contiguous epitope mimetics). The medium signal is plotted in black and the extremely high signal is plotted in light. The boxed areas are enlarged.

Antibody FAR02 VH3/VK5 and FU-HI:3-372.

When tested under high stringency conditions, the FAR02 VH3/vk5 and FU-HI-3-372 antibodies did not bind any of the peptides present in the arrays. When tested under low stringency conditions, these antibodies specifically bound peptides from group 8 only (FIG. 2). Analysis of results obtained using non-contiguous mimetics suggest that the antibody recognizes a non-contiguous epitope consisting of the sequences ₃₉₀ ATQTGRVFLQ ₃₉₉ (SEQ ID NO: 3), ₄₂₀ PFTVLVPSVSSFSSR ₄₃₄ (SEQ ID NO: 1), ₄₇₃ QEITVTFNQFTK ₄₈₄ (SEQ ID NO: 2), ₅₇₆ DSLRDGRLIYLF ₅₈₇ (SEQ ID NO: 4), ₆₁₅ SKGRILTMANQVL ₆₂₇ (SEQ ID NO: 5), where the sequences 473QEITVTFNQFTK484 (SEQ ID NO: 2) and 420PFTVLVPSVSSFSSR434 (SEQ ID NO: 1), appear to represent core epitopes. An additional weaker signal was recorded for non-contiguous mimetics containing the sequence ₃₁₃ LCVYQKPGQAFCTCR ₃₂₇ (SEQ ID NO: 6). Results obtained using simple epitope mimetics do not allow epitope determination. Fig. 2 illustrates a heat map view of the results obtained for the FU-HI-3-372 antibody in group 8 (non-contiguous epitope mimetics). The medium signal is plotted in black and the extremely high signal is plotted in light. The bordered areas are enlarged.

Conclusions.

Antibodies were tested against Pepscan peptide arrays. It was possible to identify preliminary non-contiguous epitopes for all monoclonal antibodies. Peptide sequences containing epitopes are listed in Table 1. 3. Antibodies 3-266 and AK FUMM 9-11 bind separate non-contiguous epitopes. The FAR02 VH3/VK5 and FU-HI-3-372 antibodies have generally very similar binding patterns when tested in arrays and thus have been shown to recognize the same non-contiguous epitope in the FAS1/FAS2 domains.

- 16 041532

Table 3

Discovered epitopes

Antibody Epitope sequences Domain 3-266 io3oQWLKSAGITLPADRR _1O 44 FAS 3 ₈₅₇ lharcvsqegvarcr ₈₇₁ Similar to EGF 6 435TMNASLAQQLCRQHI450 FAS 1 AK FUMM 9-11 ₈₈₅ psnpcshpdrgg ₈₉₆ Similar to EGF 6 ₁₆₆ frgsacqecqdpnrf ₁₈₀ Similar EGF 1 FARO2 VH3/VK5+FU- ₄₂ oPFTVLVPSVSSFSSR ₄ 34 FAS 1 HI-3-372 4 ₇₃ QEITVTFNQFTK ₄₈ 4 FAS 1 ₃₉₀ atqtgrvflq ₃₉₉ FAS 1 ₅₇₆ dslrdgrliylf ₅₈₇ FAS 2 ₆₁₅ skgriltmanqvl ₆₂₇ FAS 2

For visual comparison of preliminary epitopes identified for the aforementioned antibodies, the scheme depicted in FIG. 3. This circuit is adapted from FIG. 1 from Kzhyshkowska, TheScientificWorldJOURNAL (2010) 10, 2039-2053, representing the domain organization of stabilin-1 (CLEVER-1). In FIG. 3 schematically illustrates the domain organization of CLEVER-1 (ak_25-1027 as for the target sequence). The arrows show the relative positions of the identified binding motifs. Circled arrows show the positions of dominant epitope cores.

Example 2

mRNA isolation, RT-PCR and cloning.

mRNA was successfully isolated from hybridoma cells (PolyA Tract system, Promega, cat. no. Z5400). RT-PCR was performed using degenerate mouse signal sequence primer pools with a single constant region primer. The heavy chain variable region mRNA was amplified using a set of six degenerate primer pools (HA-HF), and the light chain variable region mRNA was amplified using a set of eight degenerate primer pools (kA-kG and λ A). Amplification products were prepared using the HD heavy chain primer pool and the kB, kC and kG light chain primer pools, confirming that the light chain is derived from the k cassette (FIG. 4). Each product was cloned and several clones for each were sequenced.

Using this method, a single VH sequence [SEQ ID NO: 32 (base sequence) and NO: 33 (amino acid sequence)] was identified for the HD pool, and a single functional Vk sequence [SEQ ID NO: 34 (base sequence) and NO: 35 (amino acid sequence)] was identified for the kG primer pool. The heavy chain CDRs range from base 91 to 111 (SEQ ID NO: 7), 154 to 210 (SEQ ID NO: 8), and 298 to 333 (SEQ ID NO: 9); and the light chain CDRs range from base 70 to 105 (SEQ ID NO: 10), 151 to 171 (SEQ ID NO: 11), and 268 to 294 (SEQ ID NO: 12). CDR definitions and protein sequence numbering are according to Kabat. An altered k light chain transcript normally associated with hybridoma fusion partner SP2/0 (GenBank M35669) was also identified for the kB and kC primer pools.

Fig. 4 illustrates 1% agarose gel separation of RT-PCR products for hybridoma 3-372. The gel was stained with SYBR® green (Invitrogen, cat. no. S-7567) and photographed under ultraviolet light. The size marker was a GeneRuler™ 1Kb Plus (Fermentas, p/n SM1331). Boxes show bands that were isolated for cloning and sequencing.

Example 3

Sequence analysis.

Analysis of sequences derived from hybridomas expressing 3-372 are summarized in Table 1. 4.

- 17 041532

Table 4

Sequence analysis of antibody ^3-372a

H-chain L-chain Length of CDR 1 7 ac 12 ac Length of CDR 2 16 ac 7 ac CDR 3 length 12 ac 9 ak Closest human germline sequence ¹³ IGHV2-5*10 (73%) IGKV3D-20*01 (65%) Closest human FWl ^b IGHV2-70*06 (73%) IGKV1D-17*O1 (68%) Closest human FW2 ^b IGHV2-5*09 (86%) IGKV1D-39*O1 (73%) Closest human FW3 ^b IGHV2-70*13 (72%) IGKV1D-43*O1 (78%) Closest human J ^b IGHJ1 (91%) IGKJ4 (80%)

a CDR definitions and sequence numbering are according to Kabat.

^b Germ(s) ID followed by % homology.

Structural and sequence homology analysis of the mouse 3-372 variable domain identified four framework residues in the heavy chain variable region and five framework residues in the light chain variable region that are considered critical or possibly important for antigen binding (limiting residues). Additional analysis of sequence databases revealed that human framework fragments can be found to include all of the desired limiting residues and all CDR residues, thus allowing the construction of Composite Human Antibodies™ human antibodies.

Regions V 3-372 are also noted to have some unusual features. The VH chain contains an N-glycosylation site at the start of CDR1 since residue 30 is N and 32 is S (the N-glycosylation signal is NXS or NXT, where X can be any amino acid). Due to the likely exposure of this motif on the surface of the antibody, it is likely that this site may be glycosylated. Thus, it may be advantageous (if not involved in antigen binding) to exclude this region from Composite Human Antibodies in order to avoid any future manufacturing problems. The Vk chain also contains a glycosylation site, but only in the context of the human k constant region, since the final amino acid of the Vk domain is asparagine. The murine k constant domain begins with RA, while the human k constant domain begins with RT, thus forming a glycosylation signal. Thus, Composite Human Antibodies sequences can be chosen to exclude this asparagine.

The k domain also contains an unpaired cysteine at position 47. Molecular modeling suggests that this residue may be buried in the structure and thus not be available for disulfide bonding; however, it may be a key residue for maintaining the CDR2 Vk conformation, and thus sequences for Composite Human Antibodies can be selected with and without this residue (the latter includes consensus human L at this position) to investigate its effects on binding antigen.

Example 4

Expression of the chimeric antibody.

Variable regions 3-372 were transferred to the Antitope kappa heavy chain and light chain IgG4(S241P) expression vector system. NS0 cells were transfected by electroporation and subjected to selection using methotrexate (MTX). A number of MTX-resistant colonies were identified using Fc-binding ELISA/kappa chain detection, and cell lines positive for IgG expression were serially expanded from 96-well plates to T175 flasks in media containing progressively increasing concentrations of MTX and then frozen in liquid nitrogen. At each stage, IgG expression was quantified.

Chimeric IgG4 3-372 was purified from cell culture supernatants on a Protein-A Sepharose column and quantified by OD280 nm using an extinction coefficient (Ec(0.1%)) value of 1.55 based on the predicted amino acid sequence of chimeric IgG4. Approximately 90 μg of the antibody was purified and the sample analyzed by reconstituted SDS-PAGE (FIG. 5). Bands corresponding to predicted heavy and light chain sizes were observed without evidence of any contamination; however, it was seen that the chimeric light chain,

- 18 041532 appears to be glycosylated, as evidenced by a larger apparent molecular weight than that of the mouse light chain. The heavy chain also appeared to move slower than normal, suggesting that it is also N-glycosylated; however, cleavage by glycosidases is needed to show that this is indeed the cause.

Fig. 5 illustrates a Coomassie blue stained gel after SDS-PAGE purified with Protein A of chimeric IgG4 3-372. 1 μg of the sample was applied to NuPage 4-12% Bis-Tris gel (Invitrogen, cat. no. NP0322BOX) and separated at 200 V for 30 min. Lane 1 and 4: Pre-stained protein standard (Fermentas PageRuler cat. no. SM1811). Lane 2: 1.0 μg of chimeric IgG4 antibody 3-372. Lane 3: 1.0 μg mouse antibody 3-372.

Example 5

Binding of the chimeric antibody to CLEVER-1.

Binding of NS0-derived chimeric 3-372 to CLEVER-1 was evaluated by competitive ELISA. Briefly, a 96-well Nunc Immulon maxisorp plate (Fisher, cat. no. DIS-971-030J) was coated with CLEVER-1 at 1 μg/ml in PBS (100 μl/well) overnight at 4°C, with an additional 1 h at 37°C the next morning. The wells were washed with PBS/0.1% Tween 20 and then blocked for 45 minutes at room temperature, in 1% Marvel/1% BSA/PBS.

Serial dilutions of both chimeric 3-372 and reference mouse 3-372 (5-0.078 μg/ml) were premixed with a constant concentration (0.6 μg/ml) of biotinylated mouse 3-372 antibody in 2% BSA/PBS. The blocked ELISA plate was washed as before and 100 μl of the premixed antibody was added to each well. The tablet was incubated for 1 h at room temperature. Binding of biotinylated murine 3-372 to CLEVER-1 was detected using streptavidin-HRP (Sigma, cat. no. S5512) and one-component TMB substrate solution (Invitrogen, cat. no. 00-2023). The reaction was stopped with 3M HCl, absorbance was read at 450 nm in a Dynex Technologies MRX TC II plate reader, and the chimeric 3-372 binding curve was compared to that of the control mouse antibody 3-372 (FIG. 6).

In FIG. 6 shows the binding profile of mouse and chimeric antibodies to CLEVER-1 in competition with a biotinylated mouse antibody. The curves were nearly identical, yielding IC50 values of 0.89 μg/ml for the chimeric antibody versus 0.77 μg/ml for the mouse antibody. This confirms that the correct variable region sequences have been identified and expressed in the chimeric antibody.

Example 6

Design of sequences and variants of the variable region of the Composite Human Antibody™.

Structural models of the V regions of the mouse anti-CLEVER-1 antibody were generated using the Swiss PDB and analyzed to identify important limiting amino acids in the V regions that are likely to be necessary for the binding properties of the antibody. The residues contained within the CDR (using both Kabat and Chothia definitions), together with a number of framework residues, are considered important. Both the VH and Vk sequences of the anti-Clever 1 antibody contain typical framework residues, and the CDR motifs 1, 2 and 3 are comparable to many murine antibodies. However, the present inventors have identified a potential N-linked glycosylation site in the VH (30N) sequence and an unpaired cysteine in the Vk (47C) sequence.

From the above analysis, it was concluded that it was possible to generate composite human anti-CLEVER-1 antibody sequences with a wide range of alternatives outside the CDRs, but only a narrow range of possible alternative residues within the CDR sequences. Preliminary analysis showed that appropriate sequence fragments from several human antibodies can be combined to produce CDRs similar or identical to CDRs in mouse sequences. For regions outside the CDR and flanking the CDR, a wide range of human sequence fragments have been identified as possible components of the V regions of the new Composite Human Antibody™.

Based on the above analysis, a large preliminary set of sequence fragments that can be used to generate variants of the Composite Human Antibody™ against CLEVER-1 were selected and analyzed using iTope™ technology for in silico assay of peptide binding to human MHC class II alleles ( Perry et al 2008), and using TCED™ (T cell epitope database) related to known antibody sequences of T cell epitopes (Bryson et al 2010). Sequence fragments that were identified as significant non-human germline sequences binding to human MHC class II, or that were rated as significant best matches to TCED™, were discarded. This resulted in a reduced set of fragments and their combinations were analyzed again as above to ensure that the junctions between the fragments did not contain potential T-cell epitopes. The selected fragments were then combined to obtain

- 19 041532 heavy and light chain region V sequences for synthesis. For the anti-CLEVER-1 antibody, four chains VH, VH1, VH2 were designed; VH3 and VH4 [SEQ ID NOs: 13, 15, 17 and 19 (base sequence) and NO: 14, 16, 18 and 20 (amino acid sequence), respectively], and the five chains Vk, VK1, VK2, VK3, VK4 and VK5 [SEQ ID NOs: 21, 23, 25, 27 and 29 (base sequence) and NO: 22, 24, 26, 28 and 30 (amino acid sequence), respectively]. The heavy chain CDRs of VH1, VH2, VH3, and VH4 ranged from base 91 to 111, 154 to 201, and 298 to 333; and the CDRs of the light chains VK1, VK2, VK3, VK4, and VK5 ranged from base 70 to 105, 151 to 171, and 268 to 294. CDR definitions and protein sequence numbering are according to Kabat. Of note, three of the VH chains have a potential N-linked glycosylation site removed (VH2, VH3 and VH4), and two of the Vk chains have an unpaired cysteine removed (VK4 and VK5).

Example 7

Construction of Composite Human Antibody™ variants.

All variants of the genes of the VH and Vk regions of the Composite Human Antibody™ against Clever 1 were synthesized using a series of overlapping oligonucleotides, which were hybridized, ligated and PCR amplified to obtain full-length synthetic V regions. The assembled variants were then cloned directly into the Antitope pANT expression vector system for VH chains and Vk chains of IgG4(S241P) (Fig. 7). The VH region was cloned using the MluI and HindIII sites, and the Vk region was cloned using the BssHII and BamHI restriction sites. All constructs were confirmed by sequencing.

In FIG. 7 shows a schematic of the Antitope pANT vector. The vectors for both Vh and VK contain genomic DNA fragments including introns and poly-A sequences. Expression of both chains is driven by the CMV promoter and selection (for the heavy chain vector) is made by the DHFR minigene.

Example 8

Construction, expression and purification of antibodies.

All combinations of VH and Vk chains of composite IgG4(S241P) (ie, 20 pairs in total) were stably transfected into NS0 cells by electroporation. Stable transfectants were selected using 200 nM methotrexate (MTX) (Sigma, Cat # M8407), MTX-resistant colonies for each construct were tested for IgG expression levels using an IgG4 ELISA, and lines with the best expression were selected, expanded, and frozen in liquid nitrogen . Successful transfection and selection of stable clones was achieved for all variants except for VH3/Vk3 and VH4/Vk3.

Composite anti-CLEVER-1 antibody variants were purified from cell culture supernatants on a Protein A Sepharose column (GE Healthcare, cat. no. 110034-93), buffer changed to PBS, and quantified by OD280 nm using an extinction coefficient (Ec(0 .1%)=1.55) based on the predicted amino acid sequence. Composite Human Antibody™ leading variants of the Composite Human Antibody™ were analyzed by reconstituted SDS-PAGE. Bands corresponding to the predicted VH and Vk chain sizes were observed without evidence of any contamination (FIG. 8).

Fig. 8 illustrates a Coomassie blue stained gel after SDS-PAGE of protein A purified selected antibodies. 2 μg of each sample was applied to NuPage 4-12% Bis-Tris gel (Invitrogen, cat. no. NP0322BOX) and separated at 200 V for 35 min. The size marker was a Fermentas PageRuler pre-stained protein standard (Cat. No. SM1811).

Example 9

Binding of Composite Human AntibodiesTM to CLEVER-1.

Binding derived from NS0 composite antibodies 3-372 with CLEVER-1 was assessed by competitive ELISA. Serial dilutions of both chimeric and compound 3-372 antibodies (5-0.078 μg/ml) were premixed with a constant concentration (0.6 μg/ml) of biotinylated mouse 3-372 antibody. They were incubated for 1 hour at room temperature in a 96-well Immulon maxisorp plate (Fisher, cat. no. DIS-971-030J) pre-coated with 1 μg/ml CLEVER-1. Binding of biotinylated murine 3-372 to CLEVER-1 was detected using streptavidin-HRP (Sigma, cat. no. S5512) and one-component TMB substrate solution (Invitrogen, cat. no. 002023). The reaction was stopped using 3 M HCl, absorbance was read at 450 nm in a Dynex Technologies MRX TC II plate reader, and binding curves were plotted. IC50 values for each antibody were calculated and normalized to the IC50 of the chimera that was included in each respective ELISA plate.

The IC50s obtained indicate that a number of human anti-CLEVER-1 Composite Human Anti-CLEVER-1 Antibodies™ have better binding to CLEVER-1 than chimeric 3-372. The competition data for the leading variants is shown in FIG. 9.

- 20 041532

Table 5 Binding Characterization of Anti-CLEVER-1 Composite Human anti-CLEVER-1 Antibodies™

Region IDs V Relative IC50 CH/SC 10 VH1/VK1 0.84 VH1/VK2 1.37 VH1/VK3 1.63 VH1/VK4 1.17 VH1/VK5 1.13 VH2/VK1 0.82 VH2/VK2 0.95 VH2/VK3 0.7 VH2/VK4 0.79 VH2/VK5 0.52 VH3/VK1 0.76 VH3/VK2 0.51 VH3/VK3 - VH3/VK4 0.47 VH3/VK5 0.42 VH4/VK1 1.86 VH4/VK2 0.9 VH4/VK3 - VH4/VK4 1.2 VH4/VK5 0.46

Relative IC50 was calculated by dividing the value for the test antibody by the value for the chimera assayed on the same plate.

Example 10 In Vitro Antibody Binding.

Human peripheral blood monocytes were collected from healthy donors and enriched from approximately 9 ml of peripheral blood by Ficoll gradient centrifugation. They were then seeded into 96-well low-attachment plates at a density of 1.2 x 10 ⁶ cells/well in IMDM medium supplemented with 1% human AB serum. Cells were treated with 1 μg/ml or 10 μg/ml anti-CLEVER-1 antibody 3-372 (DSM ACC2520, deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 21, 2001) or VH3/VK5 (humanized antibody against CLEVER-1 of the present invention recognizing said specific CLEVER-1 epitope) within 48 hours. HLA-DR expression was determined in CD 14 positive cells after 48 hours using an LSR Fortessa flow cytometer. Dead cells were excluded from the analysis based on a positive signal for the 7-AAD dye to determine cell viability.

Human IgG was used as a reference.

In FIG. 10A shows the results of determination of HLA-DR expression on CD 14 positive cells. HLA-DR expression on CD 14 positive cells was increased by treatment with humanized anti-CLEVER-1 VH3/VK5 antibody compared to reference human IgG.

No differences in cell viability were observed between treatments. Thus, it can be concluded that CLEVER-1-targeted antibodies do not affect monocyte survival.

Example 11 TNF-oc level measurement.

Human peripheral blood monocytes from healthy donors were collected and enriched as described in Example 10. Monocytes from 3 ml of blood treated with erythrocyte lysis buffer were allowed to adhere overnight in 6-well plates, washed once with PBS, and cultured in for 3 days with 10 μg/ml of anti-CLEVER-1 3-372 or AK-1 antibody.

The level of soluble TNF-alpha was measured in the culture medium using a commercial TNF-alpha ELISA kit (Invitrogen). The measurement results are shown in FIG. 10V. Increased secretion of TNF-alpha was noted in anti-CLEVER-1 antibody samples compared to untreated samples or AK-1 control treated samples.

Example 12 Mouse models of syngeneic malignant tumors.

Established E0771 mouse mammary carcinomas were treated with 5 mg/kg of anti-CLEVER-1 antibody (mStabl) or isotype control every 3-4 days until tumors were 1 mm ³ in size. The effect of anti-CLEVER-1 antibody treatment on the recruitment and phenotype of TAMs, various monocyte subgroups, and tumor-infiltrating leukocytes was assessed using flow cytometry.

In FIG. 11A shows TAM repolarization in E0771 syngeneic breast carcinomas following administration of an antibody that binds to CLEVER-1. TAM repolarization is measured by an increase in populations of macrophages expressing MHSPs (human HLA-DR) by flow cytometry. Each

Claims (3)

the dot represents the percentage of MHCn ^hlsh CD11b ⁺ F4/80 ⁺ TAM in one mouse. Tumors treated with anti-CLEVER-1 antibody showed a similar level of TAM (CD11b ⁺ F4/80 ⁺ ) compared to tumors after control treatment. However, the TAM population in anti-CLEVER-1 treated tumors consisted of more pro-inflammatory macrophages (Ly6CloMHCIIhi) with lower expression of the type II marker, CD206. Anti-CLEVER-1 antibody-treated TAMs secreted significantly more TNF-alpha compared to IgG-treated TAMs, as shown in FIG. 11B. Each dot represents TAMs isolated from a single mouse. Consistent with this, a decrease in the number of FoxP3+ tumor-infiltrating leukocytes was also observed. The results indicate that CLEVER-1 is a potential target for macrophage-targeted immunotherapy. Example 13 As noted in Example 1, antibodies 9-11 and 3-372 bind to separate epitopes on human CLEVER-1, and the effects of this difference on signaling in human peripheral blood monocytes are currently being investigated. In FIG. 12 illustrates that binding of CLEVER-1 to antibodies 9-11 and 3-372 mimics opposite effects on mTOR (target for the mechanism of action of rapamycin) and c-Jun signaling in human peripheral blood monocytes. In FIG. 12A shows flow cytometry analysis of 9-11 and 3-372 binding on CD14 positive human monocytes (n=2 donors, D1 and D2). In FIG. 12B shows results using the Human Phospho-Kinase Array (R&D) to measure phosphoprotein activation on CD14 positive cells (enriched by negative selection) after 10 min treatment with 20 μg/ml 9-11 and 3372. Phosphorylation signals normalized according to the respective isotype control treated cells, rat IgG2a for 9-11 and mouse IgG1 for 3-372. As shown in FIG. 12B, antibodies 9-11 and 3-372 mimic opposite effects on mTOR and c-Jun signaling in human peripheral blood monocytes. The mTOR pathway is known to regulate macrophage polarization and the immunosuppressive phenotype of macrophages is dependent on c-Jun phosphorylation, where the results show that antibody 3-372 activates macrophages to switch their phenotype from M2 macrophages to M1 macrophages. Other Preferred Embodiments. It is understood that an agent capable of binding to human CLEVER-1, such as an antibody, single chain Fv or Fab fragment(s), peptide(s), macromolecule(s), and humanized antibody or humanized single chain Fv or Fab fragment(s) and pharmaceutical compositions of the present invention can be included in the form of many embodiments, only a few of which are described in the present description. A person skilled in the art will appreciate that other embodiments exist and do not deviate from the scope of the invention. Thus, the described embodiments are illustrative and should not be construed as limiting. CLAIM 1. A humanized antibody capable of binding to the common receptor-1 of the endothelium of the lymphatic system and vascular endothelium (CLEVER-1) of a person, where the antibody contains: a) human IgG4 kappa heavy chain and light chain constant regions, and b) a combination of human IgG heavy and light chain variable regions selected from the group consisting of the following combinations: SEQ ID NO: 14 and SEQ ID NO: 22, SEQ ID NO: 16 and SEQ ID NO: 22, SEQ ID NO: 16 and SEQ ID NO: 24, SEQ ID NO: 16 and SEQ ID NO: 26, SEQ ID NO: 16 and SEQ ID NO: 28, SEQ ID NO: 16 and SEQ ID NO: 30, SEQ ID NO: 18 and SEQ ID NO: 22, SEQ ID NO: 18 and SEQ ID NO: 24, SEQ ID NO: 18 and SEQ ID NO: 28, SEQ ID NO: 18 and SEQ ID NO: 30, SEQ ID NO: 20 and SEQ ID NO: 24 and SEQ ID NO: 20 and SEQ ID NO: 30. 2. The humanized antibody of claim 1, wherein the combination of human IgG heavy and light chain variable regions is preferably selected from the group consisting of the following combinations: SEQ ID NO: 16 and SEQ ID NO: 30, SEQ ID NO: 18 and SEQ ID NO : 30 and SEQ ID NO: 20 and SEQ ID NO: 30. 3. A humanized antibody according to claim 1 or 2, wherein the constant region of the heavy chain and light chain -