WO2013160498A1

WO2013160498A1 - Human anti-her2 monoclonal antibody

Info

Publication number: WO2013160498A1
Application number: PCT/ES2013/000062
Authority: WO
Inventors: Iván SÁNCHEZ DE MELO; Enrique VILLEGAS MARTÍNEZ; Jorge BOLIVAR PÉREZ; Laura HERNÁNDEZ RUIZ; Carmen CASTRO GONZÁLES; Francisco José GARCÍA COZAR; Carlos PENDÓN MELENDEZ; Jesús Manuel CANTORAL FERNÁNDEZ; Francisco Javier FERNÁNDEZ ACERO; Carlos GARRIDO CRESPO
Original assignee: Universidad De Cádiz; Curaxys, S.L
Priority date: 2012-04-27
Filing date: 2013-03-07
Publication date: 2013-10-31
Also published as: ES2431914B2; ES2431914A1

Abstract

The present invention relates to a human monoclonal antibody that recognizes the extracellular domain of human epidermal growth factor receptor 2 (HER2) and is produced in human embryonic retinal cells. The present invention also relates to the use of said antibody to measure the level of expression of HER2 and to prepare a drug for treating neoplastic disorders involving overexpression of HER2.

Description

ANTI-HER2 HUMAN MONOCLONAL ANTIBODY

FIELD OF THE INVENTION

The present invention belongs to the field of recombinant protein production, more specifically it relates to a human monoclonal antibody that recognizes the extracellular domain of the human epidermal growth factor receptor 2 (HER2) and is produced in human embryonic retinal cells. Also, the present invention relates to the use of said antibody, in particular for the preparation of a medicament for the treatment of neoplastic disorders that occur with HER2 overexpression.

BACKGROUND OF THE INVENTION

The majority of therapeutic antibodies developed as medical agents are human lgG1 isotypes including mouse / human, humanized and human chimeric lgG1. Human IgG1 is a glycoprotein that has in most cases two N-biantena complex type glycosylations attached to the constant part (Fe) of the antibody, in which most oligosaccharides are fucosylated. Human lgG1 exerts among other functions, effector functions in the Antibody Dependent Cellular Cytotoxicity (ADCC) and in the Complementary Dependent Cytotoxicity (CDC) through the interaction of the Fe region with each one of the leukocyte receptors (FcyRs) or complement. The efficacy of therapeutic antibodies results from the specificity of the target antigen and the effector functions of the antibody, which are activated by the formation of immune complexes. It has recently been shown that the ADCC and CDC response is one of the largest anti-neoplastic mechanisms responsible for the clinical efficacy of therapeutic antibodies.

One of the common characteristics of the therapeutic antibodies used in the treatment of cancer is that its anti-tumor efficacy requires high serum concentrations and continuous therapy for several months. The treatment cycles therefore require several grams of therapeutic antibody, resulting in a significant amount of drug and very high costs. In fact, a high dose (2-8 mg kg ^"1 ) of rituximab lgG1 anti-CD20 (Rituxan ®) or trastuzumab lgG1 anti-Her2 (Herceptin ®) is required for achieve an effective serum concentration of more than 10 pg ml_ ^"1. Unlike these doses in vivo, the maximum cytotoxic activity in vitro via ADCC of the therapeutic antibodies is achieved with concentrations of less than 10 ng kg ^{~ 1.} This discrepancy between In vivo and in vitro efficacy is mainly due to the fact that serum IgG, although it does not affect the antigen binding capacity of the therapeutic antibody, hinders the binding of the therapeutic antibody with the Fc Rllla receptor of NK cells by inhibiting the ADCC response induced by said antibodies It has been found that afucosylated (non-fucosylated) therapeutic antibodies can evade said inhibitory effect of human plasma IgG on ADCC.

The majority of therapeutic antibodies currently allowed on the market are produced in rodent cell lines such as Chinese Hamster Ovary (CHO) cells, NS0 and SP2 / 0 mouse myeloma and mouse hybridoma, with almost all molecules produced by these lines. Cells fucosylated in Fe oligosaccharides, which means that the ADCC response is not optimal. In addition, in the CHO cellular system the enzymes responsible for carrying out the glycosylation processes differ from humans, as is the case of the enzyme that performs the addition of galactose (GnTI, GnTII), very active and abundant in humans unlike the mouses. In this regard, different studies have described that an increase in the percentage of galactosylation results in a better CDC response in recombinant IgGs, while such increase does not have a negative effect on the ADCC response.

The use of non-human cells for the production of recombinant proteins can also cause other problems, such as an incorrect folding of the proteins that occurs during or after translation, which may be dependent on the presence of the appropriate chaperone proteins. An aberrant folding can lead to a decrease or absence of biological activity of the recombinant protein, a decrease in the half-life in blood and even an increase in immunogenicity. In addition, simultaneous expression and in correct relative amounts of those polypeptides that make up the different subunits of quaternary structure proteins, such as antibodies, are of great importance. The antibodies consist of a basic unit composed of two heavy globular polypeptide chains and two light polypeptide chains linked together by disulfide bridges. Both chains have a constant zone and a variable zone. In the latter, there is a hypervariable zone formed by 10 to 15 amino acids responsible for specific binding with the antigen. As for therapeutic antibodies for the treatment of cancer, Trastuzumab (marketed by Roche as Herceptin®) is a humanized monoclonal antibody lgG1 that recognizes the extracellular domain of receptor 2 of human epidermal growth factor (HER2). This antibody is designed to block the proliferation pathway activated by the HER2 receptor, produced by a specific gene with carcinogenic potential. The production of said antibody is carried out using the CHO cell line, which can lead to the drawbacks described above (decreased biological activity, increased immunogenicity, etc.). Therefore, there is still a need in the state of the art to achieve a human anti-HER2 monoclonal antibody with a glycosylation pattern that favors a better immune response.

OBJECT OF THE INVENTION The present invention relates in one aspect to a polypeptide characterized in that it comprises the polypeptide sequence SEQ ID NO 1. It also refers to the nucleotide sequence encoding said sequence SEQ ID NO 1 and a vector comprising said nucleotide sequence. It also refers, in another aspect, to a human embryonic retinal cell characterized in that it has been transfected with said vector.

Another aspect of the invention relates to a human monoclonal antibody that recognizes HER2 characterized in that it is produced by said human embryonic retinal cell. And it also refers to an immunotoxin comprising a conjugate of said antibody and at least one cytotoxic agent. It also refers to a kit comprising said antibody or said immunotoxin.

In another aspect, the present invention relates to a pharmaceutical composition comprising said antibody and a pharmaceutically acceptable carrier. It also refers to a pharmaceutical composition comprising an immunotoxin comprising a conjugate of said antibody with at least one cytotoxic agent, and a pharmaceutically acceptable carrier.

The present invention relates, in another aspect, to the use of said antibody to measure the level of HER2 expression. It also refers to the use of said antibody for the preparation of a medicament for the treatment of a neoplastic disorder that occurs with HER2 overexpression. Also, it refers to the use of an immunotoxin comprising a conjugate of said antibody and at least one cytotoxic agent, for the Preparation of a medicament for the treatment of a neoplastic disorder that occurs with HER2 overexpression.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a CRX01 antibody binding assay (human anti-HER2 monoclonal antibody produced in PER.C6® cells whose heavy chains comprise SEQ ID NO 1, whose light chains comprise SEQ ID NO 4 and whose glycosylation profile comprises a percentage of afucosylation less than or equal to 4% and a percentage of galactosylation greater than or equal to 35%) and Herceptin® performed by Western Blot under reducing conditions using as primary antibody CRX01 (A) or Herceptin® (B), both at a concentration of 5 μg / ml, and as a secondary antibody anti-human IgG peroxidase at a 1: 20,000 dilution. 300 ng of a peptide corresponding to the extracellular domain of the HST2 protein conjugated to the GST protein, having a total molecular weight of 35 kDa was loaded. As a negative control, an extract of JKT cells was charged. The Western Blot shows that CRX01 is able to bind under reducing conditions to HER2, being able to block its activity.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a polypeptide comprising the amino acid sequence SEQ ID NO 1. It also refers to a DNA, cDNA or RNA molecule comprising the sequence encoding the polypeptide of SEQ ID NO 1. In A particular embodiment, said DNA molecule comprises the sequence SEQ ID NO 2.

Another aspect of the present invention relates to a vector comprising a DNA molecule encoding a polypeptide comprising the sequence SEQ ID NO 1, in a particular embodiment said vector comprises SEQ ID NO 2. In another particular embodiment said vector comprises furthermore a nucleotide sequence encoding a polypeptide of sequence SEQ ID NO 4, said said nucleotide sequence comprising, in a particular embodiment, the nucleotide sequence SEQ ID NO 5. In a particular embodiment, the vector comprises the nucleotide sequence SEQ ID NO 3. In another particular embodiment, said vector has the sequence SEQ ID NO 3. The antibodies consist of a basic unit composed of two heavy globular polypeptide chains (also referred to as heavy chains) and two light polypeptide chains (also referred to as light chains) linked together by disulfide bridges. Both chains (heavy and light) have a constant zone and a variable zone. The sequence SEQ ID NO 1 encodes each of the heavy polypeptide chains of the human anti-HER2 monoclonal antibody object of the present invention (see below) and the sequence SEQ ID NO 4 encodes each of the light polypeptide chains of said antibody. It should be noted that in the heavy chain polypeptide sequence the sequence residues 359 and 361 of the constant domain are aspartic acid (D) and leucine (L), respectively, corresponding to the G1 m17.1; Km3 allotype.

Another aspect of the present invention relates to a human embryonic retinal cell (hereinafter cell of the invention) characterized in that it is transfected with a vector comprising a DNA molecule encoding a polypeptide comprising the sequence SEQ ID NO 1. In A particular embodiment, the cell of the invention is transfected with a vector encoding a polypeptide comprising the sequence SEQ ID NO 1 and a polypeptide comprising the sequence SEQ ID NO 4. In another particular embodiment, the cell of the invention is transfected. with a vector comprising the nucleotide sequence SEQ ID NO 3. In another particular embodiment, the cell of the invention is the PER.C6® cell (Crucell, Percivia), deposited in the European Collection of Cell Cultures (ECACC) with the number 96022940 (patents ES 2333425 T3 and EP 1445322 B1 of the holder Crucell Holland BV). In a particular embodiment, the cell of the invention is ECACC cell 96022940 transfected with the vector comprising SEQ ID NO 3. In a particular embodiment, said vector is transfected after being linearized, in particular by enzymatic digestion with the restriction enzyme. Seal Said ECACC cell 96022940 transfected with the SEQ ID NO 3 sequence vector linearized with Seal is deposited according to the Budapest Treaty at the Health Protection Agency Culture Collections (HPACC) with deposit number 12030701. Another aspect of the present invention relates to a human monoclonal antibody that recognizes HER2 (hereinafter antibody of the invention) is produced by the cell of the invention. Said anti-HER2 antibody recognizes HER2 as shown in Figure 1. In the terms of the present invention it is understood as "human recombinant monoclonal antibody", also referred to as "human monoclonal antibody", all that monoclonal antibody produced in cells human through recombinant DNA technology. Production in human cells carries the important advantage that the antibodies produced have a human glycosylation profile and can therefore be less immunogenic than those produced in non-human cells. In a particular embodiment, the antibody of the invention is characterized in that it has a glycosylation profile comprising a percentage of afucosylation less than or equal to 4%. In another particular embodiment, the antibody of the invention has a glycosylation profile comprising a percentage of galactosylation greater than or equal to 35%. In another particular embodiment, the antibody of the invention has a glycosylation profile comprising a percentage of afucosylation less than or equal to 4% and a percentage of galactosylation greater than or equal to 35%, and more particularly, said percentage of afucosylation is less than or equal to 3.5% and said percentage of galactosylation is greater than or equal to 40%, and even more particularly said percentage of afucosylation is less than or equal to 3% and said percentage of galactosylation is greater than or equal to 45% . These percentages of fucosylation and galactosylation represent an important advantage since lower levels of fucosylation have been linked to a better ADCC response, while higher levels of galactosylation have been linked to a better CDC response, which allows said antibodies to be administered in doses. minors

Another aspect of the invention relates to a kit comprising the antibody of the invention as defined in the preceding paragraph. In another aspect, the present invention relates to an immunotoxin comprising a conjugate of the antibody of the invention as defined above, and at least one cytotoxic agent. The invention also referring, in another aspect, to the kit comprising said immunotoxin. Another aspect of the present invention relates to a pharmaceutical composition comprising the antibody of the invention as defined above, and a pharmaceutically acceptable carrier. It also refers to a pharmaceutical composition comprising an immunotoxin comprising an antibody conjugate of the invention as defined above and at least one cytotoxic agent, and a pharmaceutically acceptable carrier.

The present invention in another aspect relates to the use of the antibody of the invention to measure the level of expression of the HER2 protein. It also refers to the use of the antibody of the invention for the preparation of a medicament for the treatment of a neoplastic disorder that occurs with HER2 overexpression. In a particular embodiment, said neoplastic disorder that occurs with HER2 overexpression is HER2-positive breast cancer. A final aspect of the present invention relates to the use of an immunotoxin comprising a conjugate of the antibody of the invention as defined above and at least one cytotoxic agent, for the preparation of a medicament for the treatment of a neoplastic disorder in progress. with the overexpression of HER2. In a particular embodiment, said neoplastic disorder that occurs with HER2 overexpression is HER2-positive breast cancer.

BIOLOGICAL MATTER DEPOSIT

The ECACC cell line with the number 96022940 (PER.C6®) transfected with the vector v074 (SEQ ID NO 3) linearized by enzymatic digestion with Seal has been deposited with the Health Protection Agency Culture Collections (HPACC, Gate Down, Salisbury, Wiltshire, SP4 OJG, United Kingdom) on March 7, 2012, with deposit number 12030701.

Examples Specific examples of embodiments of the invention that serve to illustrate the invention are detailed below.

In the present examples, as mentioned above, CRX01 refers to an anti-HER2 human monoclonal antibody whose heavy chains comprise SEQ ID NO 1, whose light chains comprise SEQ ID NO 4, and whose glycosylation profile comprises a percentage of afucosilaclón less than or equal to 4% and a percentage of galactosylation greater than or equal to 35%.

Example 1. Generation of clones producing the CRX01 antibody Culture of PER.C6® cells

Throughout the culture process, PER.C6® cells were grown in PerMab medium (Thermo Fisher Scientific / HyClone, catalog number SH3087 .0), which is specially designed to allow high density cell growth. The PerMab medium was completed with the addition of the amino acid glutamine (Ajinomoto) 3mM and the detergent pluronic acid F-68 (Sigma, catalog number P1300) at 1% and in no case was any type of serum added to the medium. Said PerMab medium supplemented with glutamine and pluronic acid is also referred to as "complete medium" hereafter. The culture process began with the thawing of a vial of PER.C6® cells (Cruceil) and its cultivation in complete medium for a week in T-Flask 75 cm ² (Corning) under static conditions. The cells were then adapted for agitation for which they were grown in Shake Flask 250 ml (Corning) at 75 rpm in an orbital shaker.

The culture was generally under-cultivated every 3 or 4 days, sowing 0.5 x 10 ⁶ viable cells / ml, except in Batch production assays in which initially 1 x 10 ⁶ viable cells / ml were seeded.

Transfection of the PER.C6® cell line with the sequence vector SEQ ID NO 3 (vector v074).

Two days before transfection the cells were passed by centrifugation to completely change the medium and 0.7x10 ⁶ viable cells / ml were seeded. 5x10 ⁶ viable PER.C6® cells from culture in Shake Flask were transfected, with 5 pg of the expression vector v074, which encodes the heavy and light chain of the monoclonal antibody CRX01. Vector v074 was transfected in a circular and linear form (digested with the restriction enzyme Seal and purified with QIAquick columns (QIAGEN)) obtaining two stable pools of cells expressing CRX01 which were called stable pool PER.C6-CRX01 -circular and stable pool PER.C6-CRX01 -linear, respectively. Transfection was performed by electroporation using the V nucleofector kit and the AMAXA transfection apparatus with the X001 program.

After transfection, the cell density was determined and 0.5x10 ⁶ cells / ml were plated in serum-free PerMab medium supplemented with 3mM glutamine and 1% pluronic acid F-68 (complete medium) without geneticin. In addition, a negative transfection control was performed.

At 48 hours after transfection the culture was reseeded at a concentration of 0.5x10 ⁶ viable cells / ml in T-Flask by changing the medium completely and supplementing it with 3mM glutamine, 1% pluronic acid F-68 and 125 pg geneticin (G418 , Invitrogen).

From this point and for two weeks it was subcultured, sowing 0.3x10 ⁶ viable cells / ml and renewing the PerMab medium supplemented with 3mM glutamine, 1% pluronic acid F-68 and 125 pg G418.

Feasibility levels fell by up to 20% in both transfections. When the parameters of growth and viability began to improve, approximately from day 15, the crop was already considered stable. From that moment, the stable pool of cells expressing the v074 vector was subcultured every 3 or 4 days by dilution in T-Flask by sowing 0.5x10 ⁶ viable cells / ml and renewing the PerMab medium supplemented with 3mM glutamine, 1% acid pluronic F-68 and 125 pg G418. Generation of a Master Cell Bank (MCB) and a Working Cell Bank (WCB) of the stable pool Per.C6-CRX01 -linear and of the stable pool Per.C6-CRX01 -circular

When the viability exceeded 85%, an MCB of both stable pools was generated in PerMab medium supplemented with 3 mM glutamine and 1% pluronic acid F-68. This MCB was generated under static conditions.

An aliquot of the MCB from both pools was then thawed and cultivated under static conditions during the first week in PerMab medium supplemented with 3mM glutamine and 1% pluronic acid, from which the cells were adapted to stirring at 75 rpm using the same culture medium supplemented with G418. When the viability parameters were above 90% and the doubling times were between 30 and 40 hours, the WCB of both stable pools was generated.

Selection of clones producing the CRX01 antibody

To select super-producing clones of the CRX01 antibody, a limit dilution process was performed, which consisted of sowing between 0.5 and 1 cells per well in 96-well plates, from which two screenings of each of them were carried out. the stable, circular and linear pools. A first screening in 96-well plates from which the 24 most productive clones were chosen. These clones were subjected to a second T-Flask screening of 25 cm ² , from which the 4 clones that produced the most CRX01 were chosen, called PER.C6-CRX01 -Lin45, PER.C6-CRX01-Lin90, PER.C6- CRX01- Circ49, PER.C6-CRX01-Circ103.

Example 2.- Afucosylation and galactosylation of the CRX01 antibody.

A comparative study of the afucosylation and galactosylation percentage of Herceptin and CRX01 produced in PER.C6® cells has been carried out following the instructions of the PER.C6® cell distribution company (Percivia) and described in the previous example. For this, an enzyme release assay of glycans has been carried out, followed by fluorescent tide and analysis by UPLC (high efficiency liquid chromatography). The N-glycans were released from the IgG samples by overnight incubation with N-glycosidase F (Prozyme, Hayward, CA). The released glycans were recovered in the dry supernatant after precipitation of the protein with 75% ethanol. The oligosaccharides were labeled with the 2-aminobenzamide fluorophore (2AB, Prozyme) and analyzed using "Acquity UPLC® BEH glycan column" (Waters) following the manufacturer's instructions. With the data obtained, the afucosylation and galactosylation percentages were calculated according to the following formulas: G0 + G1 + G2

% afucosylation: X 100

G0 + G0F + G1 + G1F + G2 + G2F

GlF + (2 x G2F)

% galactosylation = _Λ „„ x 100

2 x (G0F + GlF + G2F)

GO, G1 and G2 refer to complex, neutral, biantena structures with 0, 1 and 2 terminal galactose residues, while GOF, G1 F and G2F are the corresponding fucosylated structures. The results obtained are shown in Table 1, where it is observed that the afucosylation percentage in CRX01 (obtained from different PER.C6-CRX01 clones both linear and circular) is less than the afucosylation percentage in Herceptin. Specifically, the afucosylation percentage in CRX01 is less than 4%, while that of Herceptin is 5%. As for the percentage of galactosylation, it is higher in CRX01 than in Herceptin, said percentage being greater than 40% in CRX01, while in Herceptin it does not exceed 25%.

Table 1.- Afucosylation and galactosylation percentage of CRX01 and Herceptin.

Claims

1. Polypeptide characterized in that it comprises the sequence SEQ ID NO 1.

2. DNA, cDNA or RNA molecule characterized in that it comprises the sequence encoding a polypeptide according to claim 1.

3. DNA molecule according to the preceding claim characterized in that it comprises the sequence SEQ ID NO 2.

4. Vector comprising a DNA molecule according to any one of claims 2 or 3.

5. Vector according to claim 4 further comprising a DNA molecule comprising a sequence encoding a sequence polypeptide SEQ ID NO 4.

6. Vector according to claim 5 comprising the nucleotide sequence SEQ ID NO 3.

7. Human embryonic retinal cell characterized in that it is transfected with a vector according to claim 4.

8. Cell according to claim 7 wherein the human embryonic retinal cell is ECAAC cell No. 96022940.

9. Human embryonic retinal cell characterized in that it has been transfected with a vector according to claim 5 or 6.

10. Cell according to claim 9 wherein the human embryonic retinal cell is ECAAC cell No. 96022940.

11. Cell according to claim 10 deposited in the HPACC with the deposit number 12030701.

12. Human monoclonal antibody that recognizes the extracellular domain of receptor 2 of human epidermal growth factor (HER2) characterized in that it is produced by a cell according to any one of claims 9 to 11.

13. Antibody according to claim 12 with a glycosylation profile comprising a percentage of afucosylation less than or equal to 4%.

14. Antibody according to claim 12 with a glycosylation profile comprising a percentage of galactosylation greater than or equal to 35%.

15. Antibody according to claim 12 with a glycosylation profile comprising a percentage of afucosylation less than or equal to 4% and a percentage of galactosylation greater than or equal to 35%.

16. Immunotoxin comprising a conjugate of an antibody according to any one of claims 12-15, and at least one cytotoxic agent.

17. Pharmaceutical composition comprising an antibody according to any one of claims 12-15 or an immunotoxin according to claim 16, and a pharmaceutically acceptable carrier.

18. Use of an antibody according to any one of claims 12-15 for the detection of the level of expression of the HER2 protein.

19. Use of an antibody according to any one of claims 12-15 or an immunotoxin according to claim 16 for the preparation of a medicament for the treatment of a neoplastic disorder that is overexpressed with HER2.

20. Use of an antibody according to claim 19 for the preparation of a medicament for the treatment of HER2-positive breast cancer.

21. Use of an immunotoxin according to claim 19 for the preparation of a medicament for the treatment of HER2-positive breast cancer.

22. Kit comprising an antibody according to any one of claims 12-15 or an immunotoxin according to claim 16.