Factor B and C2 protein point mutants, a method for enhancing the activity of anti-cancer antibodies, the pharmaceutical composition and the use of mutants

Abstract

The subject of this invention is point mutants of human proteins constituting the complement system's C3 and C5 convertases, where the mutation are as follows: For the factor B: —D279G, F286L, K323E, Y363A; D279G_F286L_K323E_Y363A—quadruple mutant; For the C2 protein: —C261A, Q.263G, Y347A, L348A; T442Q, double mutants C261A_Q263G and Y347A_Q263G, triple mutant Y347A_Q263G_T442Q The subject of this invention is the method of enhancing the activity of the anti-cancer antibodies, which includes the addition of mutants defined above. The subject of this invention is the pharmaceutical composition, which includes the therapeutically effective number of mutants defined above. The subject of this invention is the use of mutants defined above to enhance the cytotoxic activity of anti-cancer antibodies in therapy and in the treatment of neoplastic diseases.

Claims

1. A composition comprising a mutant Factor B protein, wherein the mutant Factor B protein is a Factor B protein with D279G, F286L, K323E, and Y363A mutations, wherein the mutations are relative to the Factor B protein of SEQ ID NO.:1 and wherein the composition enhances the cytotoxic activity of one or more immunotherapeutics.

2. A composition comprising a mutant C2 protein, wherein the mutant C2 protein is selected from the group consisting of: a C2 protein with C261A and Q263G mutations; a C2 protein with Q263G and Y347A mutations; and a C2 protein with Y347A, Q263G, and T442Q mutations, wherein the mutations are relative to the C2 protein of SEQ ID NO.:2, and wherein the composition enhances the cytotoxic activity of one or more immunotherapeutics.

3. A pharmaceutical composition comprising: a therapeutically effective amount of a composition of claim 1; and one or more anti-cancer antibodies.

4. The pharmaceutical composition of claim 3, wherein one or more anti-cancer antibodies are anti-CD20 antibodies.

5. A method of treating a patient having a neoplastic disease comprising: administering a therapeutically effective amount of a composition of claim 1 to a patient in need thereof in combination with one or more immunotherapeutics.

6. The method of claim 5, wherein one or more immunotherapeutics are anti-CD20 antibodies.

7. The method of claim 6, wherein the neoplastic disease is cancer.

8. A pharmaceutical composition comprising: a therapeutically effective amount of a composition of claim 2; and one or more anti-cancer antibodies.

9. The pharmaceutical composition of claim 8, wherein one or more anti-cancer antibodies are anti-CD20 antibodies.

10. A method of treating a patient having a neoplastic disease comprising: administering a therapeutically effective amount of a composition of claim 2 to a patient in need thereof in combination with one or more immunotherapeutics.

11. The method of claim 10, wherein one or more immunotherapeutics are anti-CD20 antibodies.

12. The method of claim 11, wherein the neoplastic disease is cancer.

Description

FIGURES

Description of Figures

(1) FIG. 1 demonstrates the cytotoxic effect of anti-CD20 antibodies, depending on the dose of antibodies.

(2) FIG. 2 shows the cytotoxic effect of anti-CD20 antibodies after serum supplementation with recombinant gain-of-factor B factor mutations. The NHS (normal human serum) symbol was used for the serum without the addition of factor B, the WT (wild-type) symbol for the result of wild-type (non-mutated) supplementation and to the height of this bar (dashed line) reference to the calculations of statistically significant differences were made. Factor B mutants markings refer to the position of a given amino acid in the native protein (the numbering includes the signal sequence) and changes of a specific amino acid naturally occurring in a given position (letter before the amino acid position number expressed with the so-called one-letter code) to the amino acid resulting from the mutation (letter after position number expressed using the so-called single-letter code). The 4× marking corresponds to a quadruple mutant grouping the mutations D279G, F286L, K323E and Y363A. The ANOVA test with Dunnett post-test showed statistical significance for supplementation with the factor B quadruple gain-of-function mutation in the case of Raji cells at the significance level of p<0.05 (*).

(3) FIG. 3A shows the anti-CD20 antibodies' cytotoxic effect after the serum supplementation (at the concentration optimal for visualizing the antibodies' cytotoxic effect in a given cell line—10% for the Raji cells and 20% for Namalwa cells, respectively) with C2 protein's gain-of-function recombinant mutants. Markings on the graph analogically to FIG. 2. The statistical significance according to the ANOVA test with Dunnett post-test at a level of p<0.001 is marked on the chart with a *** symbol and the significance at a level of p<0.05 with a * symbol.

(4) FIG. 3B shows overall experiment results comprising two lower, suboptimal serum concentrations for which the cytotoxic effect of the anti-CD20 antibodies in a given cell line was tested.

(5) FIG. 4 shows results of an additional experiment that compared the effect of single mutants ((Y347A, Q263G, T442Q), double mutant (Q263G_Y347A) and a triple mutant (Q263G_Y347A_T442Q)) on the Namalwa cells in an optimal serum concentration (20%, FIG. 4A) and additionally with two suboptimal serum concentrations (FIG. 4B). The markings on the charts accordingly to FIG. 2 and FIG. 3. The statistical significance according to the ANOVA test with a Dunnett post-test at a level of p<0.001 is marked on the chart with a *** symbol, and the significance at a level of p<0.05 with a ** symbol.

(6) FIG. 5 shows results of an experiment in which the effect of C2 protein supplementation of the serum taken from patients treated with rituximab anti-CD20 antibody combined with an additional amount of another anti-CD20 antibody ofatumumab was tested. The effect of supplementation with the use of the recombined wild-type C2 protein (WT) and supplementation of the C2 protein multiple mutant (Q263G_Y347A_T442Q) were compared. The experiment was carried out with a 50% patient serum concentration (which reflected the physiological conditions) on the Namalwa cell line characterized by a low level of sensitivity to anti-CD20 antibodies. The ANOVA test with a Dunnett post-test showed a statistical significance to the multiple C2 protein gain-of-function mutant's supplementation in relation to a supplementation with unmutated (WT) protein at a significance level of p<0.01 (**).

(7) FIG. 6 shows results of an experiment in which the 50% serum taken from patients after the rituximab administration (and thus containing some amount of rituximab unused by the immune system) was supplemented with a C2 protein multiple mutant (Q263G_Y347A_T442Q) or an unmutated protein (WT) and then was used in a cytotoxic test on Raji cells. The ANOVA test with a Dunnett post-test showed a statistical significance for supplementation with the multiple C2 protein gain-of-function mutant (causing a complete lysis of the Raji cells by the patients' sera) in relation to the supplementation of unmutated protein (WT) at a significance level of p<0.001 (***).

(8) FIG. 7 shows Seq.1, which means a factor B's sequence with a signal sequence (single-letter amino acid code).

(9) FIG. 8 shows Seq.2, which means a C2 protein's sequence with a signal sequence (single-letter amino acid code). The invention is illustrated by, but not limited to, the following examples:

EXAMPLE 1

(10) Description of Methodology

(11) The cDNA reference sequences of the C2 proteins (accession number NM_000063.5) and the factor B (accession number NM_001710), additionally containing at the end 3′ triplets for six histidine residues, have been optimized in terms of the GC pairs content, the presence of unfavorable secondary structures and the so-called rare codons through the ThermoFisher Scientific algorithm. The optimized sequences were then synthesized de novo and cloned to the pCEP4 expression plasmid. The constructs were once again verified with the help of the DNA sequencing. Plasmids verified for the sequence correctness were transformed into the DHSα strain E. coli bacteria, from which the plasmid DNA was purified with the help of MidiPrep Kit (Qiagen) in the amount necessary for the eukaryotic cells' transfection. With the help of 30 μg of plasmid DNA and the Freestyle Max (ThermoFisher) reagent, a transfection of the HEK 293 Freestyle cells was performed. The cells were grown for seven days in the Freestyle 293 Expression Medium (ThermoFisher), while the growth medium was harvested on the second, fourth and seventh day after the transfection and resupplied with a fresh medium. The harvested, cell-conditioned medium was stored at −80 C until the protein purification process with the use of affinity chromatography. For this purpose, HisTrap FF crude (GE Healthcare) columns were used. Washing and balancing the columns were performed in a 20 mM Tris-HCl pH 8.0 buffer with addition of 1 mM of imidazole. The elution was conducted with the use of 0.7 M imidazole in the same buffer. The protein containing eluate fractions were merged, dialyzed on the PBS buffer and concentrated with the use of Vivaspin mwco. 10 kDa (Millipore) concentrator. On the basis of the cDNA optimized matrix, the cDNAs of factor B's single mutants D279G, F286L, K323E, and Y363A were created and the multiple mutant containing all of the substitutes mentioned above (4X), whereby the protein expression of a single F286L mutant was not achieved. Similarly, complement C2 protein single mutants (R243C, C261A, Q263G, S307E, Y347A, L348A, and T442Q) were created successively, as well as double mutants C261A_Q263G and Y347A_Q263G and the triple mutant Y347A_Q263G_T442Q.

(12) The human Raji and Namalwa lymphoma cells expressing the CD20 marker on their surface were used for experiments. The Raji cells incubated in the presence of human serum are characterized by an average sensitivity to anti-CD20 antibodies, the maximum achievable level of cells' lysis oscillates within 50%. The Namalwa cells are characterized by a low sensitivity to the anti-CD20 antibodies due to the less favorable ratio of the CD20 molecules to the amount of the complement system's surface inhibitors, such as CD46, CD55 and CD59. Depending on the experiment, the Namalwa cells' lysis level in the presence of the serum and the anti-CD20 antibodies oscillated between 10% and 20%. The impact of the recombined factor B and the complement system's C2 protein on the anticancer activity of the anti-CD20 antibodies was tested in an in vitro cytotoxic test. Cells suspended in the RPMI medium with a 10% bovine serum addition were incubated with a 1 mM calcein derivative (calcein-AM), which is actively collected by living cells and metabolized to a fluorescent derivative. The incubation was carried out at 37° C. in a 5% CO2 environment for 30 minutes. The cells were then washed three times with a PBS buffer, suspended in a PBS buffer supplemented with a 1 mM CaCl.sub.2 and MgCl.sub.2 and added in the amount of 100.000 to the well of a V-shaped multiwell plate. After centrifugation and supernatant's removal, the cell pellet was suspended in human serum with anti-CD20 antibody (ofatumumab) in a standard concentration of 50 μg/ml. For the Raji cells, a 10% serum was used as a concentration for optimal visualization of the anti-CD20 antibodies' cytotoxic effect, for the Namalwa cells analogically the concentration was increased to 20%. Additionally, in selected experiments, a 50% serum collected from patients suffering from B cell-derived tumors treated with rituximab therapeutic anticancer antibody was used. The final reaction volume was 50 μl. The cells were incubated with the serum and with anti-CD20 antibodies for 30 minutes in a thermomixer (Eppendorf) at 37° C. and with shaking at 650 rpm. After the incubation was finished, another 50 μl of the PBS buffer was added and then the plate with the cells was centrifuged at a speed of 1000×G for two minutes. Eighty μl of the supernatant was moved to the wells of a 96-well flat-bottomed plate and the fluorescence of the collected supernatants was measured at excitation/emission wavelengths of 485 and 515 nm, respectively. The intensity of the fluorescence was directly proportional to the fluorescence of the derivative released from the cells due to the lysis induced by the complement system contained in the serum. The cells incubated with the heat-inactivated serum (56° C., 30 min) and thus lacking the complement's activity were treated as a negative control, i.e., a lysis independent of the complement system's activity.

(13) In the first experiment it was shown that the increase in the dose of the anti-CD20 antibodies at a constant serum concentration does not significantly increase the percentage of the cells destroyed. (FIG. 1)

(14) The bar marked with a Δ 500 symbol illustrated the effect of the maximum antibody concentration (500 μg/ml) and a heat-inactivated serum. Its height illustrates the level of lysis of cancer cells independent from the complement system. The ANOVA test with a Dunnett post-test did not show statistically significant differences between the 50 μg/ml concentration and the higher concentrations.

(15) In contrast to increasing the antibodies' concentration, increasing the cytotoxic effect was possible after the serum (NHS) supplementation with factor B gain-of-function-type mutants (FIG. 2) and analogical complement's C2 protein mutants (FIG. 3). The concentration of the used mutants was equal to the physiological concentration of the proteins in the serum at a given concentration. For a 10% serum they were 2.5 μg/ml C2 and 20 μg/ml factor B and 5 μg/ml C2 protein for a 20% serum.

(16) In the case of C2 protein, a panel of single gain-of-function mutants, two double mutants and one triple mutant, were made. The best effects (statistically significant increase in the cell's lysis percentage) in experiments performed in the Raji cells with single and double mutants were obtained for a Y347A_Q263G mutant—ANOVA test with a Dunnett post-test showed significance at a level of p<0.001 in the Raji cells case. A single Y347A mutant was at a similar level. It should be noted, however, that a noticeable effect of the Y347A_Q263G double mutant increasing the cytotoxicity of the anti-CD20 antibodies was already present at a suboptimal 5% serum concentration (FIG. 3B). In the case of Namalwa cells, statistically significant differences in the wild-type C2 protein supplementation were found in Q263G, T442Q, Y347A, C261A_Q263G, Y347A_Q263G and Y347A_Q263G_T442Q mutants (separate experiment depicted in FIG. 4). The R243C mutant was treated as a negative control due to the mutation's characteristic disrupting the structure, and hence, the function of the C2 protein. Double mutants, whose efficacy on the Namalwa line was demonstrated at a 20% serum's concentration, noticeably increased the cytotoxic effect also in a suboptimal 10% serum's concentration (FIG. 3B). The obtained results suggest that the multiple mutants work more efficiently under conditions of other complement proteins' deficiency, which can reflect an in vivo situation, in which the complement's pool is depleted due to earlier immunotherapy rounds or a primary or secondary immunodeficiency.

(17) In a separate experiment on Namalwa cells the anti-CD20 antibodies' cytotoxic effect with supplementation with single (Y347A, Q263G, T442Q), double (Q263G_Y347A) and triple (Q263G_Y347A_T442Q) C2 protein mutants was compared (FIG. 4). The aim of the experiment was to show that the accumulation of single C2 protein gain-of-function-type mutations in one protein construct does not lead to the loss of C2 protein activity but may cause an additive effect manifested by both a higher antibodies' cytotoxicity at an optimal serum concentration and a significantly higher percentage of killed cells at a suboptimal serum concentration, in comparison to the results obtained under analogical conditions for single mutants. Such actions are desirable from a practical point of view because it ensures a much more beneficial supplementation effect.

(18) In the next experiment, the Namalwa line cells along with sera (in the final 50% concentration corresponding to the physiological conditions) taken from the patients with B cell-derived tumors treated with rituximab were used. Serum samples were taken from patients directly before and directly after the administration of rituximab. The rituximab's administration causes a mass activation of the complement system in this type of patients, which in turn is manifested by a decreased serum's cytotoxic activity after the drug administration, due to the consumption of the complement protein pool. A sustained state of decreased cytotoxic activity may cause a decreased rituximab's efficacy in relation to cancer cells that have not been killed during the first administration of the drug. The sera were first supplemented with another anti-CD20 antibody, analogical to rituximab: ofatumumab. Next, the sera were supplemented with a recombinant wild-type C2 protein and Q263G_Y347A_T442Q multiple C2 protein mutant at a concentration of 12.5 μg/ml. The cytotoxic test results showed that supplementing the patients' sera with a multiple mutant can not only increase the cytotoxic effect of the patient before the drug administration but can also counteract the reduction of the cytotoxic activity of patients' sera following the rituximab's administration at the time when the drug's activity is leading to exhaustion of the complement system's protein pool (FIG. 5). The described multiple mutant's effect could not be obtained by a supplementation with a recombinant, wild-type (WT) C2 protein variant.

(19) An analogical experiment, in which the effects of the wild-type (WT) and triple-mutated (Q263G_Y347A_T442Q) C2 protein supplementation on the cytotoxic potential of the sera collected from the same patients as in FIG. 5, although without the addition of saturating concentration of ofatumumab, was carried out on the Raji cell line (FIG. 6). In this experimental setup, the only present anticancer drug was the excess rituximab remaining after the intravenous administration, which was not used by the immune system to the cancer cell elimination. The addition of 12.5 μg/ml of a triple-mutated C2 protein allowed for a complete lysis of the Raji cells by sera collected from patients after the rituximab's infusion, whereas the result for the sera without the C2 addition or with an addition of an unmutated (WT) C2 protein ranged between 50% and 70% of a complete lysis depending on the patient. The obtained results illustrate the example, when not the bioavailability of the drug, but the decrease in the serum's cytotoxic activity is the factor limiting a successful anticancer antibody therapy and when the of gain-of-function C2 protein mutants can significantly increase the effectiveness of the drug already present in the patient's bloodstream by strengthening the complement system's activity.

(20) Results Summary:

(21) It has been shown that some gain-of-function-type mutations in the C2 proteins and in the factor B can significantly increase the cytotoxic activity of the anti-CD20 antibodies such as ofatumumab, for example. An analogical supplementation effect is to be expected for other anticancer antibodies known from literature (e.g., rituximab, alemtuzumab) acting via the complement system. It has been shown, however, that a similar effect cannot be reproduced by increasing the dose of antibodies alone. An accumulation of many point mutations in one protein may give a cumulative effect, as in the case of a quadruple factor B D279G_F286L_K323E_Y363A mutant or the C2 protein Y347A_Q263G, C261A_Q263G, Y347A_Q263G_T442Q mutants. The use of multiple C2 protein mutants such as Q263G_Y347A_T442Q was able to counteract the decrease in the cytotoxic potential of the sera collected from the patients after the anti-CD20 antibodies administration and also to significantly better utilize the anticancer potential of the antibodies remaining in the patients' sera after the drug administration. As an element of novelty, we emphasize the use of such mutants as a universal supplement in antibodies anticancer immunotherapy.

LITERATURE

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