MODEL FOR PREDICTION OF TOLERABILITY ISSUES IN CONNECTION WITH INTRAVENOUS ADMINISTRATION OF THERAPEUTIC ANTIBODIES

Abstract

The present invention generally relates to combinations for use in therapeutic systems and antibody dosage regimens, and uses thereof. Also described herein is a model for predicting if a therapeutic antibody binding to a human target will be associated with a tolerability issue in connection with intravenous administration and/or for predicting if pre-treatment, altered administration route or modification of the antibody can prevent a tolerability issue associated with intravenous administration to a human of the therapeutic antibody. The model comprises administering the antibody intravenously or intraperitoneally to mice and observing the mice immediately after the administration for any transient display of the macroscopic symptoms isolation and decreased activity. The model may also comprise administration of a pre-treatment in combination with administration of the antibody, administration of the therapeutic antibody by a route of administration other than intravenous or intraperitoneal administration or administration of a modified format of the antibody to mice and observing the mice immediately after such administration for any transient display of the macroscopic symptoms isolation and decreased activity and comparing this with the transient display of the macroscopic symptoms isolation and decreased activity after the intravenous or intraperitoneal administration of the unmodified antibody without pre-treatment.

Claims

1. A method for predicting if a therapeutic antibody molecule binding specifically to a human target will be associated with a tolerability issue in connection with intravenous administration to a human, comprising the following step: (i) intravenous or intraperitoneal administration of the therapeutic antibody molecule, if cross-reactive with murine target, or a surrogate antibody, to a mouse and observation of the mouse during a period following immediately after the administration of the therapeutic or surrogate antibody, wherein a display of the macroscopic symptoms isolation and decreased activity during the period followed by restoration of the state of the mouse to the normal state is an indication that the intravenous administration of the therapeutic antibody molecule to a human will be associated with a tolerability issue, and/or for predicting if a prophylactic or therapeutic treatment, an altered administration route and/or a modification of the therapeutic antibody molecule can prevent or mitigate a tolerability issue associated with intravenous administration to a human of a therapeutic antibody molecule binding specifically to a human target, comprising the following step(s) in addition to (i) as set out above: (ii) administration of a prophylactic or therapeutic agent to a mouse in conjunction with intravenous or intraperitoneal administration of the therapeutic or surrogate antibody to a mouse, and observation of the mouse during a period following immediately after the administration of the therapeutic or surrogate antibody, wherein a decreased display of the macroscopic symptoms compared to the macroscopic symptoms displayed by the mouse in (i) or no display of the macroscopic symptoms during the period is an indication that pre-treatment with the prophylactic or therapeutic agent in combination with administration of the therapeutic antibody molecule to a human can prevent or mitigate the tolerability issue that otherwise would be associated with intravenous administration of the therapeutic antibody molecule to a human; (iii) administration of the therapeutic or surrogate antibody to a mouse by a route of administration other than intravenous or intraperitoneal administration, and observation of the mouse during a period following immediately after the administration of the therapeutic or surrogate antibody, wherein a decreased display of the macroscopic symptoms compared to the macroscopic symptoms displayed by the mouse in (i) or no display of the macroscopic symptoms during the period is an indication that the other route of administration can be used for administration of the therapeutic antibody molecule to a human to prevent or mitigate the tolerability issue that would be associated with intravenous administration of the therapeutic antibody molecule to a human; and/or (iv) intravenous or intraperitoneal administration of a modified format of the therapeutic or surrogate antibody to a mouse by a route of administration other than intravenous or intraperitoneal administration, and observation of the mouse during a period following immediately after the administration of the modified therapeutic or surrogate antibody, wherein a decreased display of the macroscopic symptoms compared to the macroscopic symptoms displayed by the mouse in (i) or no display of the macroscopic symptoms during the period is an indication that administration of the therapeutic antibody molecule in the modified format to a human can be used to prevent or mitigate the tolerability issue that would be associated with intravenous administration of the therapeutic antibody molecule to a human.

2. A method according to claim 1, wherein a display in (i) of 1-3 additional macroscopic symptoms selected from impaired balance, piloerection, and hunching followed by un-natural body posture during the period in (i) where after the state of the mouse is restored to the normal state further strengthens the indication that the intravenous administration of the therapeutic antibody molecule to the human will be associated with a tolerability issue.

3. A method according to claim 1, wherein the period during which the macroscopic symptoms are displayed in (i) starts 5-10 minutes after administration of the therapeutic or surrogate antibody and ends 45-90 minutes after administration of the therapeutic or surrogate antibody, and wherein the observation period in (ii), (iii) and/or (iv) is of the same length.

4. A method according to claim 1, wherein at least one of the following additional parameters: decreased blood pressure decreased platelet count, and or increased liver enzymes (AST/ALT). observed during the period in (i) further strengthens the indication that the intravenous administration of the therapeutic antibody molecule to a human will be associated with a tolerability issue.

5. A method for predicting if a prophylactic or therapeutic treatment can prevent or mitigate a tolerability issue associated with intravenous administration to a human of a therapeutic antibody molecule binding specifically to a human target according to claim 1 comprising at least steps (i) and (ii), wherein pre-treatment is used in (ii) and wherein this pre-treatment is administration of a corticosteroid to the mouse prior to injection of the therapeutic or surrogate antibody.

6. A method according to claim 5, wherein the pre-treatment comprises two administrations of a corticosteroid, wherein one is given 10.sup.−48 hours prior to administration of the therapeutic or surrogate antibody and the other is given 5 minutes-5 hours prior to administration of the therapeutic or surrogate antibody.

7. A method according to claim 6, wherein the corticosteroid is dexamethasone or betamethasone.

8. A method for predicting if an altered administration route can prevent or mitigate a tolerability issue associated with intravenous administration to a human of a therapeutic antibody molecule binding specifically to a human target according to claim 1 comprising at least steps (i) and (iii), wherein the route of administration used in (iii) is subcutaneous administration.

9. A method for predicting if a modification of the therapeutic antibody molecule can prevent or mitigate a tolerability issue associated with intravenous administration to a human of a therapeutic antibody molecule binding specifically to a human target according to claim 1 comprising at least steps (i) and (iv), wherein the modified format of the therapeutic or surrogate antibody used in (iv) is a modification that leads to decreased or abolished engagement of Fc receptors.

10. A method according to claim 1, wherein the human target is selected from the group consisting of FcγRIIB, FcγRIIA and CD40.

11. A method according to claim 10, wherein the therapeutic antibody molecule is a human anti-FcγRIIB antibody capable of binding a human FcγR via its Fc domain and wherein the mouse surrogate antibody is an anti-FcγRIIb antibody capable of binding a mouse FcγR via its Fc domain, such as wherein the therapeutic antibody molecule is a human anti-FcγRIIB IgG1 antibody and wherein the mouse surrogate antibody is an anti-FcγRIIb mIgG2a.

12. (canceled)

13. A method of preventing or mitigating a tolerability issue in connection with intravenous administration of a therapeutic antibody molecule to a subject comprising administering to said subject a corticosteroid, wherein the therapeutic antibody molecule has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of claim 1, and wherein the dosing regimen comprises administration of the corticosteroid to the subject in at least two doses prior to intravenous administration of the therapeutic antibody molecule, wherein one dose of the corticosteroid is administered 10-48 hours prior to start of the administration of therapeutic antibody molecule (“the first dose”) and one dose of the corticosteroid is administered 5 minutes-5 hours prior to the start of administration of the therapeutic antibody molecule (“the second dose”).

14. A method according to claim 13, wherein the therapeutic antibody is an Fc receptor binding antibody such as an anti-FcγRIIB antibody.

15. (canceled)

16. A method of preventing or mitigating a tolerability issue in connection with intravenous administration of a therapeutic antibody molecule to a subject comprising administering to the subject a corticosteroid, wherein the therapeutic antibody molecule is an anti-FcγRIIB antibody, and wherein the dosing regimen comprises administration of the corticosteroid to the subject in at least two doses prior to intravenous administration of the therapeutic antibody molecule, wherein one dose of the corticosteroid is administered 10-48 hours prior to start of the administration of therapeutic antibody molecule (“the first dose”) and one dose of the corticosteroid is administered 5 minutes-5 hours prior to the start of administration of the therapeutic antibody molecule (“the second dose”).

17. A method according to claim 13, wherein the first dose is given 6-36 hours prior to start of administration of the therapeutic antibody molecule and the second dose is given 15-120 minutes prior to start of administration of the therapeutic antibody molecule.

18. A method according to claim 13, wherein the first dose is given 16-24 hours prior to start of administration of the therapeutic antibody molecule, and/or the second dose is given 30-60 minutes prior to start of administration of the therapeutic antibody molecule.

19. (canceled)

20. A method according to claim 13, wherein the dosing regimen comprises administration of the at least two doses of the corticosteroid prior to each infusion of the antibody during the course of antibody therapy.

21. A method according to claim 13, wherein the corticosteroid is dexamethasone or betamethasone or a combination of dexamethasone and betamethasone.

22. A method according to claim 13, wherein the corticosteroid is dexamethasone and wherein the first dose is 4-20 mg and the second dose is 4-25 mg, or wherein the first dose is 10-12 mg and the second dose is 20 mg.

23. (canceled)

24. A method according to claim 13, wherein the corticosteroid is betamethasone and wherein the first dose is 3.2-16 mg and the second dose is 3.2-20 mg, or wherein the first dose is 8-9.6 mg and the second dose is 16 mg.

25. (canceled)

26. A method according to claim 13, wherein the dosing regimen further comprises administration of an antihistamine 10 minutes −24 hours prior to start of administration of the therapeutic antibody molecule.

27. A method according to claim 14, wherein the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2.

28. A method of treating cancer in a subject comprising subcutaneously administering to the subject a therapeutic antibody molecule, wherein the therapeutic antibody molecule has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of claim 1.

29. A method according to claim 28, wherein the therapeutic antibody is an anti-FcγRIIB antibody, such as wherein the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2.

30. (canceled)

31. A method of treating cancer in a subject comprising administering to said subject a therapeutic antibody molecule, wherein the therapeutic antibody molecule has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of claim 1, and wherein the therapeutic antibody molecule is an Fc receptor binding antibody and the modified format is an antibody having the same Fv variable sequence but having impaired or abrogated FcγR binding compared with the therapeutic antibody molecule.

32. A method according to claim 31, wherein the therapeutic antibody is an anti-FcγRIIB antibody, optionally as wherein the modified format of the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 295.

33. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0409] FIG. 1: A mouse model that recapitulates BI-1206 tolerability profile. When the murine surrogate anti-CD32b antibody (AT-130-2 IgG2a) is injected into wildtype C57/BL6 mice intravenously (i.v.) or intraperitoneally (i.p.) the mice display reactions that recapitulates the BI-1206 tolerability profile in the clinic. A. shows the % of mice displaying macroscopic IRRs such as, isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture after injection. When titrating the i.v. dose the same timing and severity of macroscopic symptoms is seen down to 10 mg (0.5 mg/kg). However, at 4 mg (0.2 mg/kg) no IRRs were seen. When administrating 200 mg (10 mg/kg) i.p. a delay in IRRs onset was seen in comparison with i.v. injection with the IRRs appearing 20-30 minutes post injection. When increasing the i.p. dose to 400 mg (20 mg/kg) the onset of IRRs was still delayed compared to the i.v. injection route however, all mice displayed IRRs to the same extent and grade as 200 mg i.v. All mice had fully recovered 1 h post injection. B. PLT (platelet counts in blood) were analysed in fresh blood using a Vetscan shows that mice displaying IRRs (gray bars) also display a decrease in platelet counts. C. Blood samples were also analyzed for AST showing an increase in the group receiving 200 ug anti-CD32b antibody i.v. D. shows IL6 levels in blood of mice injected i.p. with 200 ug anti-CD32b antibody over time post injection. A peak is seen 1 h post injection and levels are back to normal 8 h post injection (gray area). A similar pattern was seen for IL-5, IL-10, KC/GRO, TNF-α.

[0410] FIG. 2: Pre-medication with two doses of corticosteroids dose-dependently block or reduce IRRs in vivo. Mice were either pretreated with: A 40 mg/kg or B 10 mg/kg Betamethasone 24 h and 1 h (premed) pre i.v. injection of 10 mg/kg AT-130-2 IgG2a. Mice were bled 20 minutes post injection. The blood was analyzed for platelet counts. Premedication with 10 mg/kg Betamethasone did not completely inhibited IRRs or decrease in platelet count B (striped bars) indicating that lowering the dose of premedication might reduce its potential to inhibit IRRs and platelet decreases.

[0411] FIG. 3: Split dose—a small pre-dose of Ab lowers the severity of IRRs and platelet decrease. A. Split-dosing was initiated with 8 ug/mouse of mouse anti-CD32B AT-130-2 IgG2a i.v. followed by a bulk-dose of 200 ug/mouse 1 h later. In parallel mice were injected with only the bulk-dose. IRRs were studied (and visualized in the figure according to the grading system in B) and platelet counts, and body temperature was measured and compared. A small pre-dose of AT-130 (8 ug) lowers the severity/protects against IRR's, platelet decrease, and body temperature decrease when giving a large/main dose (200 ug). Gray area indicates normal range of PLT (platelet counts in blood) and body temperature. The pre-dose needs to be 8 ug (a dose where IRRs are seen in 50% of the mice C), lower doses are not protective.

[0412] FIG. 4: Combined pre-medication with steroids and antibody split-dosing is needed for full (antibody) tolerance and protection against IRRs. Split-dosing was initiated either: A 24 h post suboptimal corticosteroid treatment (10 mg/kg) or B without corticosteroid pretreatment with 8 ug/mouse of mouse anti-CD32B AT-130-2 IgG2a i.v. followed by a bulk-dose of 200 ug/mouse 1 h later. In parallel, mice were injected with only the bulk-dose. IRRs were studied and platelet counts, and body temperature was measured and compared. The small pre-dose of AT-130 (8 ug) is well tolerated if a “low” dose of corticosteroids (10 mg/kg) is given 24 h before. The suboptimal pre-med together with the small pre-dose of AT-130 (8 ug) fully protects against IRR's and platelet decrease associated with a large Ab dose (200 ug). Suboptimal dose corticosteroids (10 mg/kg) is not protective itself. Gray area indicates normal range of PLT and body temperature.

[0413] FIG. 5: Pre-medication with several different clinically relevant substances does not inhibit IRRs associated with AT-130-2 IgG2a administration. In order to evaluate if pre-treatment with substances generally used in the clinic to treat IRRs could inhibit IRRs in this model, mice were pre-treated with anti-PAF, anti-IL6, anti-histamine or with a leukotriene-antagonist. These pre-medications were given i.p 1 h prior to injection i.v of mouse anti-CD32B AT-130-2 IgG2a as a bulk dose of 200 ug/mouse. Mice were observed for macroscopic IRRs such as isolation, mobility, and fur condition. None of these pre-medications could inhibit IRRs.

[0414] FIG. 6: Tolerability profile seen in human subjects with non-hodgkins B-cell lymphoma after administration of BI-1206 at doses of 70-100 mg. (A) Platelet decrease following treatment with BI-1206. The decrease is transient and most episodes have been resolved within a week and never serious or associated with bleeding. Each dot denotes a measurement and the line median. Vertical striped line indicates BI-1206 administration. (B) Platelet decrease is associated with ALT elevations. Though ALT/AST elevations have only been significant in 3/14 patients. (C) Frequency of detected cytokine elevations following treatment with BI-1206. A transient cytokine release has been detected in 7 of 8 subjects where serum/plasma has been available for analysis. The peak cytokine release is seen immediately after infusion and always gone within 24 h. (D) Kinetics of platelet decrease, ALT and cytokine elevations is exemplified by individual 504-001. Cytokines are here exemplified by IL-6.

[0415] FIG. 7: FcgRIIb receptor occupancy translates into B cell depletion. Clinical data is in line with pre-clinical data from in vivo models using hFcgRIIB transgenic mice where it has been demonstrated that a sustained receptor saturation is necessary to achieve sustained B lymphocyte depletion. Two doses of corticosteroids prior to administration anti-FcgRIIb mAb and a small pre-dose of mAb (split dose) improves tolerability (IRRs). FcgRIIb receptor occupancy on peripheral B cells (A) and peripheral B cell levels (B) in human subjects with non-hodgkins B-cell lymphoma treated with 100 mg BI-1206 monotherapy. FcgRIIb receptor occupancy (E) and peripheral B cell depletion (F) in hFcgRIIB transgenic mice after i.v. administration of increasing doses of BI-1206. (C) Grade of infusion related reactions (IRRs) in human subjects with non-hodgkins B-cell lymphoma treated with 70-100 mg BI-1206. Dark grey bars with black symbols denotes administrations without pre-medication with two doses of corticosteroids; light grey bars and open white symbols denotes administrations with pre-medication with two doses of corticosteroids. On the X-axis subject id and antibody dose is indicated. (D) Significantly lower frequency and severity of IRRs are seen after implementation of premedication with two doses of corticosteroids in the clinical protocol; black symbols dosing without two doses of corticosteroids, grey symbols dosing with two doses of corticosteroids; P<0.0001 using Mann Whitney U-test. (G) Infusion related reactions after i.v. administration of anti-FcgRIIb mAb (AT-130-2 mIgG2a) in wild type mice. Dose titration shows that at 0.4 mg/kg Ab IRRs appear in approximately 50% of the animals and at i.v. administrated doses above IRRs are seen in 100% of the animals. Pre-medication with two 40 mg/kg doses of betapred completely block IRRs whereas two 40 mg/kg doses of betapred partly block IRRs. Adding split dos to the suboptimal 10 mg/kg betapred blocks IRRs. The split dose is a small pre-dose of 0.4 mg/kg Ab followed by a large dose of 10 mg/kg Ab. (H) Improved tolerability and sustained efficacy is seen after implementation of premedication with two doses of corticosteroids in the clinical protocol. CR: complete response; PR: partial response; SD: stable disease; DLT: dose limiting toxicity.

[0416] FIG. 8. Transient FcγRIIB receptor occupancy and peripheral lymphocyte depletion after BI-1206 infusion. FIG. 8A) FcγRIIB receptor occupancy on peripheral B cells after BI-1206 infusion followed over time in subjects having received 100 mg BI-1206 (n=8). Vertical dashed lines denote BI-1206 infusions. At second and third BI-1206 infusion receptor occupancy data is only available pre-infusion. Line denotes the median. Based on preliminary data. FIG. 8 B) Peripheral lymphocytes after BI-1206 infusion followed over time in subjects having received 100 mg BI-1206 (n=9). Vertical dashed lines denote BI-1206 infusions. Line denotes the median. Based on preliminary data.

[0417] FIG. 9. Transient decrease of platelets associates with increase in ALT after BI-1206 infusion. FIG. 9A) Platelet count after BI-1206 infusion followed over time in subjects having received 70-100 mg BI-1206 (n=16). Vertical dashed lines denote BI-1206 infusions. Line denotes the median. Based on preliminary data. FIG. 9 B) Fold increase of ALT versus percent platelet depletion (n=16). Changes are calculated in relation to day 1, pre-BI-1206-infusion. Based on preliminary data.

[0418] FIG. 10. Cytokine release after BI-1206 infusion. Number of patients with plasma/serum increase of different cytokines detected at end of BI-1206 infusion. To be considered as positive the value should be >10-fold increased as compared to pre-infusion and >10-fold above the upper limit of normal range (ULN). Samples for analysis have been available from 5 subjects receiving 70 mg BI-1206. Based on preliminary data.

[0419] FIG. 11. Two doses of dexamethasone relieved IRRs, platelet decrease, and transaminase increase in two subjects receiving BI-1206. Platelet count and ALT in subjects 501-001 (FIG. 11A) and 503-002 (FIG. 11 B). Vertical dashed lines denote antibody infusions. The first infusion in each subject was rituximab alone and the following BI-1206 and rituximab. Grade of IRR at each antibody infusion is indicated. 501-001 and 503-002 received 12 mg and 4 mg dexamethasone, respectively, the evening before and again 20 mg dexamethasone 30 minutes prior to the third administration of BI-1206 (70 mg) during induction therapy. None of the subjects suffered any IRRs after this premedication regimen with two doses of dexamethasone. During the previous two infusions with BI-1206, where dexamethasone (20 mg) was given only 30 minutes prior to infusion, IRRs (grade 2-3) had been experienced. In addition, in subject 501-001 and 503-002 no/low platelet decrease, and no ALT/AST increase was seen after BI-1206 administration when premedicating with two doses of dexamethasone.

[0420] FIG. 12. Macroscopic symptoms after injection of murine surrogate anti-CD32b (AT-130-2 IgG2a). The murine surrogate anti-CD32b (AT-130-2 IgG2a) was injected into wildtype C57/BL6 mice through 3 different injection routes, intravenously i.v., intraperitoneally i.p. or subcutaneously s.c. Macroscopic symptoms were seen after i.v. and i.p. injection. These macroscopic symptoms included isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture and the macroscopic symptoms were scored from 0-2 based on the observations. When titrating the i.v. dose a rapid onset of IRRs was seen 5-7 minutes post injection. The same timing and severity of macroscopic symptoms was seen down to 10 μg (0.5 mg/kg). However, at 1 μg (0.05 mg/kg) no macroscopic symptoms were seen. When administrating 200 μg (10 mg/kg) i.p. a delay in onset of macroscopic symptoms was seen in comparison with i.v. injection with the macroscopic symptoms appearing 20-30 minutes post injection. In contrast to the i.v. injection route all mice in this group did not display macroscopic symptoms and the macroscopic symptoms were less severe in several mice. When increasing the i.p. dose to 400 μg (20 mg/kg) the onset of macroscopic symptoms was still delayed compared to the i.v. injection route however, all mice displayed macroscopic symptoms to the same extent and grade as 200 μg i.v. All mice had fully recovered 1 hour post injection. Finally, when administrating 200 μg to mice s.c., no macroscopic symptoms were seen (up to 24 hours post injection). When increasing the s.c. dose to 400 μg the mice remained unaffected.

[0421] FIG. 13. Pharmacokinetic profiles of AT-130-2 IgG2a in C57BL/6 mice. Observed AT-130-2 serum concentrations in mice treated with AT-130 via three different administrations routes; 200 μg AT-130-2 via i.v. injection, 200 μg AT-130-2 via i.p. injection and 400 μg AT-130-2 via s.c. injection. Presented data are the mean of 1-4 mice/dose groups. Abbreviations: h=hour(s); i.p. intraperitoneal, i.v. intravenous, s.c. subcutaneous. Note that complete and sustained FcgRIIB receptor is achieved with following s.c. dosing of AT130, although s.c. C.sub.Max is lower and kinetics to obtain full saturation is slower compared with i.v. or i.p. dosing.

[0422] FIG. 14. Correlation between macroscopic symptoms (denoted IRRs in this figure) and high, rapid exposure of AT-130-2 rather than time of FcγRIIB saturation. The serum concentration of AT-130-2 IgG2a (line with dots) is plotted against the grade of macroscopic symptoms (line with squares) for the different administration routes (A: i.v., B: i.p. and C: s.c.). When comparing the serum concentration of AT-130-2 and the presumed receptor occupancy (RO) (dotted line, 10 mg/ml gives 100% receptor saturation) with the onset, severity and duration of IRRs it is clear that there is a correlation between high and rapid exposure of AT-130-2, rather than time of FcγRIIB saturation. Tolerability showing a clear pattern of s.c. >i.p. >i.v. with RO being sustained for a long period of time post recovery from macroscopic symptoms.

[0423] FIG. 15A. Timing of platelet (PLT) nadir is correlated to the route of administration and time to FcγRIIB saturation. Mice were bled at different time points post injection of AT-130-2 IgG2a and the blood was analyzed for blood platelet count (PLT) in an automated Vetcount. A nadir in platelet count was seen at the same time as onset of macroscopic symptoms after injection of AT-130-2 through both the i.v. and i.p. administration route. For the s.c. administration route where no macroscopic symptoms are seen, a moderate drop was seen 10 hours post injection correlating with the time to FcγRIIB saturation according to PK. In all cases the PLT decrease was transient and was restored to values in the normal range within 8 hours post injection. Mice presenting macroscopic symptoms are shown with filled bars.

[0424] FIG. 15B shows that transaminase increase following injection of AT-130-2 IgG2a is circumvented when using the s.c. administration route. Mice were bled post injection of AT-130-2 IgG2a and the blood was analyzed for transaminases. For the i.v. administration route an increase is seen in transaminases (AST) 1 h post onset of macroscopic symptoms (previously established as time point for a peak value in transaminases). For the s.c. administration route where no macroscopic symptoms are seen, no increase in transaminases was seen 11 hours post injection (1 h post the time of FcγRIIB saturation according to PK (10 h)).

[0425] FIG. 16: Transient platelet decrease, transaminase increase and cytokine release, after administration of AT-130-2 IgG2a. Mice were bled at different time points after i.p. injection of 200 μg AT-130-2 and the blood was analyzed for platelet counts (FIG. 16A), transaminases (FIG. 16 B) and cytokines (FIG. 16 C). A transient PLT decrease was seen with the PLT counts fully restored 8 hours post injection (FIG. 16A). An increase in transaminases (AST and ALT) with a peak 1 h post injection was the only clinical chemistry parameter affected by AT-130-2 injection (FIG. 16 B). These increases were just like the PLT decrease transient. The same transient increase was seen when AT-130-2 was injected i.v. and no increase in transaminases was detected when AT-130-2 was injected s.c. (data not shown). A panel of cytokines including the analytes IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO, TNF-α were analyzed at different time points post injection of 200 mg i.p. Of the analyzed cytokines IL-5, IL-6, IL-10, KC/GRO, TNF-α showed a transient increase with them all except for IL-5 peaking 1-3 hours post injection (FIG. 16 C). IL-5 showed a delayed peak 3-8 hours post injection (FIG. 16 C). These are the same cytokines that have been indicated to increase in some patients in the clinical studies with BI-1206.

[0426] FIG. 17. Premedication with 2 doses of corticosteroids inhibits macroscopic symptoms associated with AT-130-2 IgG2a administration. Mice were either pretreated with 40 mg/kg betamethasone 24 hours and 1 hour or left untreated pre i.v. injection of 10 mg/kg AT-130-2 IgG2a. Mice were observed for macroscopic symptoms (FIG. 17A) and bleed 20 minutes post injection. The blood was analyzed for (FIG. 17 B) platelet counts, (FIG. 17 C) transaminases and cytokines. Premedication with betamethasone completely inhibited the macroscopic symptoms (FIG. 17A), decrease in platelet count (FIG. 17 B) and reduced the transaminases increase (FIG. 17 C). The transient cytokine release seen after i.v. injection of AT-130-2 was also inhibited by the premedication (data not shown). The same results were seen when using dexamethasone (data not shown).

[0427] FIG. 18. The dose of premedication is of importance. Mice were either pretreated with 40 mg/kg or 10 mg/kg Betamethasone 24 hours and 1 hour (premed) pre i.v. injection of 10 mg/kg AT-130-2 IgG2a. Mice were observed for macroscopic symptoms (FIG. 18A) and bleed 20 minutes post injection. The blood was analyzed for (FIG. 18 B) platelet counts. Premedication with 10 mg/kg Betamethasone did not completely inhibited macroscopic symptoms (FIG. 18A) or decrease in platelet count (FIG. 18 B) indicating that lowering the dose of premedication might reduce its potential to inhibit macroscopic symptoms and platelet decreases.

[0428] FIG. 19. Steroid premedication 1 hour prior to infusion is not sufficient to inhibit IRRs. Mice were either pretreated with 40 mg/kg Betamethasone 24 hours, 1 hour or 24 hours+1 hour pre i.v. injection of 10 mg/kg AT-130-2 IgG2a. Mice were observed for macroscopic symptoms (FIG. 19A) and bleed 10.sup.−20 minutes post injection. The blood was analyzed for platelet counts (FIG. 19 B). Single premedication with betamethasone 1 hour post injection of AT-130-2 did not inhibited macroscopic symptoms (FIG. 19A) or platelet decrease (FIG. 19 B) and single premedication with betamethasone 24 hours post injection of AT-130-2 did only reduce the macroscopic symptoms to a grade 1. This indicates that two doses of steroid treatment are needed to completely inhibit tolerability issues.

[0429] FIG. 20. Premedication with anti-histamine is not enough to inhibit tolerability issues associated with AT-130-2 IgG2a administration. Mice were either pretreated with anti-histamine alone or with 40 mg/kg Betamethasone (24 hours and 1 hour)+/−anti-histamine pre i.v. injection of 10 mg/kg AT-130-2 IgG2a. Mice were observed for macroscopic symptoms (FIG. 20A) and bleed approximately 20 minutes post injection. The blood was analyzed for platelet counts (FIG. 20 B). Premedication with anti-histamine alone did not inhibit macroscopic symptoms (FIG. 20A) but did seem to improve decrease in platelet counts (FIG. 20 B). The addition of anti-histamine to 40 mg/kg Betamethasone (24 hours and 1 hour) pre i.v. injection of 10 mg/kg AT-130-2 IgG2a did not affect macroscopic symptoms or platelet counts. The same results were seen when using three different types of anti-histamines (Zyrlex, Zantac or Au, data not shown).

[0430] FIG. 21 shows that several but not all murine surrogate antibodies induce IRRs. The murine surrogates anti-CD32b antibody (AT-130-2 mIgG2a), anti-CSFR1 (AFS98 rIgG2a), anti-EGFR (7A7 mIgG2a), anti-CD40 (FGK4.5 rIgG2a) and anti-FcγRIII (AT154-2 mIgG2a) were injected into wildtype C57/BL6 mice intravenously i.v. IRRs was seen after injection of anti-CD32b, anti-CD40 and anti-FcγRIII. The IRRs included isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture and the IRRs were scored from 0-2 based on the observations. No IRRs were seen for anti-EGFR or anti-CFSR1. When mice were pretreated with 40 mg/kg betamethasone 24 h and 1 h pre-injection of anti-CD32b or anti-CD40 no IRRs were seen (anti-FcγRIII was not evaluated with premedication). Indicating that premedication can inhibit IRRs related to different antibodies and targets.

[0431] FIG. 22 shows that antibodies inducing IRRs also induce platelet decrease. The murine surrogates anti-CD32b antibody (AT-130-2 mIgG2a), anti-CD40 (FGK4.5 rIgG2a) and anti-FcγRIII (AT154-2 mIgG2a) were injected into wildtype C57/BL6 mice intravenously i.v. Platelet counts were analyzed in fresh blood 20 min post injection using a Vetscan (Vetscan HM5 Abaxis, Triolab). A decrease in platelet counts was seen after injection of anti-CD32b, anti-CD40 and anti-FcγRIII. When mice were pretreated with 40 mg/kg betamethasone 24 h and 1 h pre-injection of anti-CD32b or anti-CD40 no platelet decrease were seen (anti-FcγRIII was not evaluated with premedication). Indicating that premedication can inhibit platelet decrease related to different antibodies and targets.

[0432] Specific, non-limiting examples which embody certain aspects of the invention will now be described. These examples should be read together with the brief description of the drawings provided above.

EXAMPLES

Example 1

Summary

[0433] A split dosing regimen in combination with corticosteroid pre-treatment was evaluated in an in vivo model recapitulating the tolerability profile seen with BI-1206 using the BI-1206 murine surrogate AT-130-2 IgG2a. The split dosing regimen in combination with corticosteroid pre-treatment improves the tolerability profile of anti-FCyRIIb treatment. Macroscopic IRRs, and platelet counts are improved with split dosing. The time span between the first dose and the second dose does not seem to be of importance while the correct timing of pre-treatment with corticosteroids appears to be important for complete tolerance of the first dose.

Materials & Methods

Test Substance

[0434] The anti-mouse CD32B IgG2a clone AT130-2 was transiently expressed in HEK293 cells. The specificity of the purified research batch was demonstrated in a luminescence-based ELISA or in FACS analyses. Endotoxin-levels of antibodies were found to be <0.1 IU/mL as determined by the LAL-Amoebocyte test.

TABLE-US-00006 Antibody clone Description Reference AT-130-2 IgG2a Mouse surrogate of mIgG2aK-AT130 ref: uct, BI-1206 2019 Jun. 7, 1443: 65

Mice

[0435] Six to eight weeks-old (17-20 g) female C57/BL6 and Balb C mice were obtained from Taconic or Janvier. Mice were injected i.v with mouse anti-CD32B AT-130-2 IgG2a either as a bulk dose of 200 ug/mouse or as a split-dose with 8 ug/mouse followed by 200 ug/mouse.

Premedication

[0436] For the corticosteroid treatment, Betapred (betamethasone, VNR: 008938, Alfasigma S.P.A.) was used at 10 mg/kg which is a suboptimal dose in these mouse models.

Split-Dosing

[0437] Split-dosing was initiated 24 h post corticosteroid treatment with 8 ug/mouse of mouse anti-CD32B AT-130-2 IgG2a i.v. followed by a bulk-dose of 200 ug/mouse 20-40 minutes later. In parallel mice were injected with only the bulk-dose.

Animal Monitoring

[0438] Mice were monitored post injection with regards to changes in behavior and macroscopic symptoms such as isolation, mobility, and fur condition. Macroscopic IRRs scoring system of 0-2 was set up based on the observations.

TABLE-US-00007 Scoring Macroscopic symptoms 0 No visible symptoms 1 Isolation, decreased activity 2 Isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture

Body Temperature

[0439] Body temperature was measured 20 min post injection of bulk dose with a mouse thermometer.

Blood Sampling

[0440] Blood samples were collected from vena saphena 20 min post injection of bulk dose of anti-CD32B for instant blood count analysis. For liver enzyme and cytokine analysis, the mice were bled from the aorta under isoflurane anesthesia just prior to sacrifice. Samples for liver enzymes and cytokines were collected 1 h and respectively 3 h after bulk dose.

Platelet Count

[0441] Platelet counts were analyzed in fresh blood using a Vetscan (Vetscan HM5 Abaxis, Triolab).

Transaminases

[0442] Transaminases were analyzed shipping of frozen serum samples to (IDEXX BioResearch Vet Med Labor GmbH).

Results and Discussion

[0443] A split dosing regimen in combination with corticosteroid pre-treatment improves the tolerability profile of anti-CD32b treatment. The tolerability profile of anti-CD32b treatment alone can be seen in FIG. 1. Macroscopic IRRs and platelet counts are improved with split dosing in combination with an initial dose of corticosteroid (FIGS. 2, 3 and 4). This was evaluated in an in vivo model recapitulating the tolerability profile seen with BI-1206 using the BI-1206 murine surrogate AT-130-2 IgG2a. The time span between the first dose and the second dose does not seem to be of importance however, the correct timing of pre-treatment with corticosteroids appears important for complete tolerance of the first dose.

Example 2

Summary

[0444] In order to evaluate if pre-treatment with other substances (aside from corticosteroids) generally used in the clinic to treat IRRs could inhibit IRRs in this model, the inventors pre-treated mice with several other clinically relevant substances. None of the tested pre-medications could inhibit IRRs in this model, suggesting they are not useful in preventing adverse effects associated with BI-1206 administration.

Materials and Methods

Test Substance

[0445] The anti-mouse CD32B IgG2a clone AT130-2 was transiently expressed in HEK293 cells. The specificity of the purified research batch was demonstrated in a luminescence-based ELISA or in FACS analyses. Endotoxin-levels of antibodies were found to be <0.1 IU/mL as determined by the LAL-Amoebocyte test.

TABLE-US-00008 Antibody clone Description Reference AT-130-2 IgG2a Mouse surrogate of mIgG2aK-AT130 ref: uct, BI-1206 2019 Jun. 7, 1443: 65

Mice

[0446] Six to eight week-old (17-20 g) female C57/BL6 and Balb C mice were obtained from Taconic or Janvier. Mice were injected i.v with mouse anti-CD32B AT-130-2 IgG2a either as a bulk dose of 200 ug/mouse or as a split-dose with 8 ug/mouse followed by 200 ug/mouse.

Premedication

[0447] For the corticosteroid treatment, Betapred (betamethasone, VNR: 008938, Alfasigma S.P.A.) was used at 10 mg/kg which is a suboptimal dose in these mouse models.

[0448] Other premedications evaluated were anti-PAF (CV3988, sc-201015, Santa Cruz 20 mg/kg), anti-IL6 (clone 15A7, BE0047, Bioxcell, 10 mg/kg), anti-histamine (Zantac, VNR: 077875, GlaxoSmithKline AB, 5 mg/kg) or a leukotriene-antagonist (131064, Apoex, 4 mg/kg). These premedications were given i.p 1 h prior to injection i.v of mouse anti-CD32B AT-130-2 IgG2a as a bulk dose of 200 ug/mouse.

Split-Dosing

[0449] Split-dosing was initiated 24 h post corticosteroid treatment with 8 ug/mouse of mouse anti-CD32B AT-130-2 IgG2a i.v followed by a bulk-dose of 200 ug/mouse 20-40 minutes later. In parallel mice were injected with only the bulk-dose.

Animal Monitoring

[0450] Mice were monitored post injection with regards to changes in behavior and macroscopic symptoms such as isolation, mobility, and fur condition. A macroscopic IRR scoring system of 0-2 was set up based on the observations.

TABLE-US-00009 Scoring Macroscopic symptoms 0 No visible symptoms 1 Isolation, decreased activity 2 Isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture

Body Temperature

[0451] Body temperature was measured 20 minutes post injection of bulk dose with a mouse thermometer.

Blood Sampling

[0452] Blood samples were collected from vena saphena 20 minutes post injection of bulk dose of anti-CD32B for instant blood count analysis. For liver enzyme and cytokine analysis, the mice were bled from the aorta under isoflurane anesthesia just prior to sacrifice.

[0453] Samples for liver enzymes and cytokines were collected 1 h and respectively 3 h after bulk dose.

Platelet Count

[0454] Platelet counts were analyzed in fresh blood using a Vetscan (Vetscan HM5 Abaxis, Triolab).

Transaminases

[0455] Transaminases were analysed by shipping of frozen serum samples to (IDEXX BioResearch Vet Med Labor GmbH).

Results and Discussion

[0456] As shown in FIG. 5, none of the tested substances (anti-PAF, anti-IL-6, anti-histamine and a leukotrien-antagonist) could prevent the IRRs associated with AT-130-2 administration in this mouse model. This suggests that only corticosteroids as a pre-treatment are capable of providing the protective effect described in Example 1. This finding is surprising considering all of these substances are generally used in clinic to treat IRRs associated with other therapeutic antibodies.

Example 3

Summary

[0457] It is apparent that iv administration of BI-1206 is frequently associated with IRRs, thrombocytopenia, transient spikes in cytokines, and less frequently but in the most severe cases, increases in liver enzymes (FIG. 6). It it therefore advantageous if a dosage regimen can be used which can prevent or mitigate these adverse effects. It is also apparent that in non-Hodgkin lymphoma patients, FcγRIIb receptor occupancy translates to B cell depletion (which correlates with in vivo data from mouse models), and sustained receptor saturation is therefore important for sustained B lymphocyte depletion, however achieving such sustained high receptor occupancy required for therapeutic benefit by i.v. injection is associated with high levels of IRRs (FIG. 7).

Materials and Methods

[0458] Platelet counts, ALT concentrations and IRR grading Platelet counts, ALT concentrations and IRR grading were obtained from clinical sites where analyzed and reported according local standard procedures. All described data from the clinical studies is preliminary, only partially quality controlled and should be considered as illustrative of the pharmacodynamic effects and tolerability associated with BI-1206.

FcgRIIb Receptor Occupancy

[0459] The FcgRIIb receptor occupancy in humans and hFcgRIIb transgenic mice was analyzed using flow cytometry. Whole blood from was incubated with either 005-005 antibody (targeting hFcgRIIb) or anti-hCD32-AF647 antibody. 005-005 binds the same epitope as BI-1206 but with much lower affinity. In the analysis, the Geo Mean of respectively mAb (005-005 and anti-human CD32) was acquired on the CD19+ cell population. Receptor occupancy (RO) was calculated using the following equations: RO (%)=((Total Receptors-Normalized Free Receptors)*100)/Total Receptors. All replicates of 005-005 geo mean of CD19+ cells were then multiplied by the Normalization factor.

Cytokine Analysis

[0460] For cytokine concentrations, frozen plasma samples were thawed and diluted ×2 and x8. Two parallel set of cytokines were analyzed, the proinflammatory assay with IL-6, IL-8, TNF-α, IFN-γ, IL-10, IL-2 and IL-4 (MesoScale Discovery (MSD) #K15049), and the chemokine assay with MIP-113, IL-113, IL-23, IL-12p70, TARC and VEGF (MSD #K15067). The assays followed the manufacturer's protocol as outlined briefly: 50 μL of sample and calibration standard were added to the appropriate MSD plates and incubated. Following washing, 25 μL of SULFO-TAG detection antibody mixture were added to each well of the corresponding plate. The plates were analyzed on a QuickPlex SQ120 Reader instrument (MSD) and cytokine concentration was calculated using the MSD software (Discovery Workbench, 2013; version LSR-4-0-12).

B Cell Depletion in hFcgRIIb Transgenic Mice

[0461] B cell depletion in hFcgRIIb transgenic mice was analyzed using flow cytometry using commercially available antibodies.

Results and Discussion

[0462] As shown in FIG. 6 it is apparent that iv administration of BI-1206 is frequently associated with IRRs, thrombocytopenia, transient spikes in cytokines, and less frequently but in the most severe cases, increases in liver enzymes. As shown in FIG. 7, achieving such sustained high receptor occupancy is required for therapeutic benefit is associated with high levels of IRRs.

Example 4

[0463] In Example 4A and Example 4B, an antibody denoted BI-1206 is used. This antibody has the following light and heavy chains:

TABLE-US-00010 Light chain: (SEQ. ID. No: 1) QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLI YADDHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCASWDDSQRAV IFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS Heavy chain: (SEQ. ID. No: 2) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWMAV ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREL YDAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0464] A modified format of BI-1206 is format wherein the glycosylation site at N297 (marked in bold above) is mutated to a Q (marked in bold below), i.e. an N297Q mutation, resulting in the following heavy chain:

TABLE-US-00011 (SEQ. ID. No: 195) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWMAV ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREL YDAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0465] As surrogate antibody and control antibody, the anti-mouse CD32B antibody AT130-2 as IgG2a isotype and the control antibody AT130-2 N297A as IgG1 isotype are used below. AT130-2 as IgG2a isotype is commercially available, for example from ThermoFisher Scientific as Catalog #12-0321-82, however #12-0321-82 is a PE conjugate so the antibody then should be modified so that it is not a conjugate. AT130-2 N297A as IgG1 isotype may be produced by any known method including substituting N in position 297 (as identified above) with A.

Example 4A—Target: FcvRIIB

Background

[0466] BioInvent International AB has developed the therapeutic monoclonal antibody BI-1206 with anti-tumor activity that can be used as single therapy or in combination with anti-CD20 targeting therapeutics or other clinically validated checkpoints inhibitors. BI-1206 binds with high specificity to CD32B (FcγRIIB) and is currently evaluated in two clinical phase I/IIa studies, CRUKD/16/001 and 17-BI-1206-02, treating patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphoma (B-cell NHL). All below described data from the clinical studies is preliminary, only partially quality controlled and should be considered as illustrative of the pharmacodynamic effects and tolerability associated with BI-1206. Part of the data is based on personal communication with individual investigators.

[0467] To date up to 100 mg of BI-1206 have been administrated, as monotherapy or in combination with rituximab, to 24 human subjects. 100 mg BI-1206 shows transient receptor saturation on peripheral B cells with 100%, or close to 100%, receptor occupancy for up to 48 hours (FIG. 8). Correspondingly a transient depletion of peripheral B lymphocytes is seen, recovered within approximately 7 days (FIG. 8). This is in line with pre-clinical in vivo models using hFcγRIIB mice where it has been demonstrated that a sustained receptor saturation is necessary to achieve sustained B lymphocyte depletion.

[0468] Frequent infusion related reactions (IRRs) have been seen during BI-1206 infusions in human subjects (FIG. 7A). Administration of ≥50 mg BI-1206 is also associated with a transient decrease in platelets (FIG. 9). Thrombocytopenia has not been serious nor associated with bleeding and most episodes resolved within a week. There appears to be a connection between platelet decrease and elevated transaminases (i.e. alanine transaminase (ALT) and aspartate transaminase (AST)), where the ALT and AST increase has been significant in 3 out of 16 subjects receiving ≥70 mg BI-1206 (FIG. 9).

[0469] Moreover, a transient cytokine release has been observed in 5 out 5 subjects receiving ≥70 mg BI-1206 where plasma or serum has been available for analysis. The cytokine release includes macrophage inflammatory protein (MIP)-1β, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-8, IL-6, and IL-4, and the peak is seen immediately after infusion and cytokines are always normalized within 24 hours (FIG. 10). The cytokine release has not been associated with clinical symptoms.

[0470] In the clinical study 17-BI-1206-02, 16 subjects have received 70-100 mg BI-1206, altogether there have been 58 BI-1206 administrations at these dose levels. 46 of these administrations were given following implementation of the in vivo protective corticosteroid-based premedication regimen identified in the animal model, (FIGS. 7C and 7H). Implementation of the in vivo animal model identified premedication regimen into the clinic resulted in statistically significant reduced severity and frequency of IRRs in human cancer patients (FIGS. 7D and 7H).

[0471] Subjects 501-001 and 503-002 received 12 mg and 4 mg dexamethasone, respectively, the evening before and again 20 mg dexamethasone 30 minutes prior to the third administration of BI-1206 (70 mg) during induction therapy. Both subjects did not suffer IRRs after this premedication regimen using two doses of dexamethasone. During the previous two infusions with BI-1206, where dexamethasone (20 mg) was given only 30 minutes prior to infusion, IRRs (grade 2-3) had been experienced. In addition, in subject 501-001 and 503-002 no/low platelet decrease, and no ALT/AST increase was seen after BI-1206 administration when premedicating with two doses of dexamethasone (FIG. 11). The third subject (201-003) had received 9 administrations of 30 mg of BI-1206 (4 doses during induction phase and 5 doses during maintenance phase) and repeatedly experienced IRR's. At the 10th BI-1206 administration premedication with two doses of dexamethasone was used, and IRR's were improved to grade 1. In subject 201-003 which received a lower dose of BI-1206 (30 mg) platelet decrease or ALT/AST increase had never been seen. Importantly, and consistent with FcgRIIB receptor saturation determining therapeutic efficacy, therapeutic efficacy was maintained following implementation of the premedication regimen in the clinic. Both complete and partial responses have been observed in patients following incorporation of the premedication regimen into the clinical protocol (FIG. 7H).

Materials & Methods

Test and Control Substances

[0472] The anti-mouse CD32B IgG2a clone AT130-2 and the control antibody (AT130-2 N297A) were transiently expressed in HEK293 cells. The specificity of the purified research batches was demonstrated in a luminescence-based enzyme linked immunosorbent assay (ELISA) or in flow cytometry analyses. Endotoxin-levels of antibodies were found to be <0.1 IU/mL as determined by the LAL-Amoebocyte test.

TABLE-US-00012 Antibody clone Description AT-130-2 IgG2a Mouse surrogate of BI-1206 as described above AT-130-2 IgG1 Fc null version of mouse surrogate of BI-1206 N297A as described above

Mice

[0473] Six to eight weeks-old (17-20 g) female C57/BL6 mice were obtained from Taconic. Mice were injected either intra-venous (i.v.), intra-perinatal (i.p.) or sub-cutaneous (s.c.) with mouse anti-CD32B AT-130-2 IgG2a in doses ranging from 1 μg-400 μg/mouse.

Premedication

[0474] For the corticosteroid treatment, Betapred (betamethasone, VNR: 008938, Alfasigma S.P.A.) or Dexamethasone (Cat. No: S1322, batch no: 02, Selleckchem) was used. For the anti-histamine treatment Zyrlex (10 mg/ml, VNR: 523084, MACURE PHARMA ApS), Zantac (25 mg/ml, VNR: 077875, GlaxoSmithKline AB) or Aeurius (0.5 mg/ml, VNR: 097288, Merck Sharp & Dohme BV) was used.

Animal Monitoring

[0475] Mice were monitored post injection with regard to changes in behavior and macroscopic symptoms such as isolation, mobility, and fur condition. Macroscopic IRRs scoring system of 0-2 was set up based on the observations:

TABLE-US-00013 Scoring Macroscopic symptoms 0 No visible symptoms 1 Isolation, decreased activity 2 Isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture

Blood Sampling

[0476] Blood samples were collected from vena saphena for instant blood count analysis. For serum concentrations of AT130-2, liver enzyme and cytokine analysis, the mice were bled from the aorta under isoflurane anesthesia just prior to sacrifice.

Serum Concentrations of AT130-2

[0477] Serum concentrations of AT130-2 mAb has been was quantified using a sandwich ELISA. Briefly, recombinant CD32B protein (Sino Biological #50030-M08H) was used as coating. Diluted samples were added to the ELISA plate, and following incubation and washing steps, detection was conducted via an HRP conjugated polyclonal donkey-anti-mouse-IgG Ab (Jackson #715-035-151). Subsequently the Pico Chemiluminescent Substrate (ThermoFisher #37069) were used and plate reading was performed with a Tecan Ultra Microplate reader.

Platelet Count

[0478] Platelet counts were analyzed in fresh blood using a Vetscan (Vetscan HM5 Abaxis, Triolab).

Transaminases

[0479] Transaminases were analyzed shipping of frozen serum samples to (IDEXX BioResearch Vet Med Labor GmbH).

Cytokines

[0480] To study the potential contributors of infusion-related reactions (IRRs) in the mice, cytokine release has been evaluated at selected timepoints in association with i.p. injection of AT-130-2 mAb. Serum samples frozen once were thawed and diluted ×2 or ×4. Cytokines were analyzed with the V-plex Proinflammatory Panel 1 Mouse kit (MesoScale Discovery #K15048D), including the analytes interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO, tumor necrosis factor (TNF)-α. The assay followed the manufacturer's protocol as outlined briefly: 50 μL of sample and calibration standard were added to the MSD plates and incubated. Following washing, 25 μL of SULFO-TAG detection antibody mixture were added to each well of the corresponding plate. The plates were analyzed on a QuickPlex SQ120 Reader instrument (MSD) and cytokine concentration was calculated using the MSD software (Discovery Workbench, 2013; version LSR-4-0-12).

Results

[0481] Macroscopic Symptoms after Murine Surrogate Anti-CD32b IgG2a (AT-130-2)

[0482] The murine surrogate anti-CD32b (AT-130-2 IgG2a) was injected into wildtype C57/BL6 mice through 3 different injection routes, intravenously (i.v.), intraperitoneally (i.p.) or subcutaneously (s.c.). At 200 μg (corresponding to 10 mg/kg) a rapid onset of infusion-related reactions (IRRs) was seen 5-7 minutes after i.v. injection. These IRRs included isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture. Blood sampling of these mice indicated reduced blood pressure. 10-15 minutes post IRRs onset these mice started to recover and 1 h post injection no macroscopic symptoms were seen.

[0483] When titrating the i.v. dose, the same timing and severity of macroscopic symptoms was seen down to 10 μg (0.5 mg/kg). However, at 1 μg (0.05 mg/kg) no IRRs were seen (FIG. 12).

[0484] When administrating the same dose 200 μg (10 mg/kg) i.p. a delay in IRRs onset was seen with the IRRs appearing 20-30 minutes post injection. In contrast to the i.v. injection route all mice in this group did not display IRRs and the IRRs were less severe in several mice (FIG. 12).

[0485] When increasing the i.p. dose to 400 μg (20 mg/kg) the onset of IRRs was still delayed compared to the i.v. injection route however, all mice displayed IRRs to the same extent and grade as 200 μg i.v. (FIG. 12). All mice had fully recovered 1 h post injection.

[0486] Finally, when administrating 200 μg to mice s.c. no IRRs were seen (up to 24 h post injection). When increasing the s.c. dose to 400 μg the mice remained unaffected (FIG. 12).

[0487] When administrating the Fc-null version of AT-130-2, AT-130-2 IgG1 N297A i.v. no IRRs were seen, indicating that Fc-binding is necessary to incite the symptoms associated with AT-130-2.

[0488] The pharmacokinetic profiles of AT-130-2 was assessed for i.v., i.p., and s.c. injection (FIG. 13).

[0489] When comparing the PK and the presumed receptor occupancy (RO, based on separate experiments not shown here where it was shown that 10 μg/ml gives 100% receptor saturation) with the onset, severity and duration of IRRs it is clear that there is a correlation between high and rapid exposure of AT-130-2, rather than time of FcγRIIB saturation. Tolerability showing a clear pattern of s.c. >i.p. >i.v. with RO being sustained for a long period of time post IRRs recovery (FIG. 14).

Platelets, Transaminases and Cytokines

[0490] To investigate if the IRRs seen in these mice were associated with other parameters seen in the clinical studies with BI-1206 mice were bled at the onset of IRRs and the blood was analyzed for blood cell count, clinical chemistry parameters and cytokines. In the case of s.c. injection where no IRRs occurred, mice were bled at different timepoints post injection. A decrease in platelet count (PLT) was seen at the same time as IRRs onset after injection of AT-130-2 through both the i.v. and i.p. administration route (FIG. 15A). For the s.c. administration route only a moderate drop was seen 10 h post injection (FIG. 15A). In all cases the PLT decrease was transient and was restored to values in the normal range within 8 h post injection (data for 200 μg AT-130-2 injected i.p. is shown in FIG. 16A). When administering AT-130-2 IgG2a s.c. the transaminase increase seen following i.v. injection is circumvented. For the i.v. administration route an increase is seen in transaminases (AST) 1 h post onset of macroscopic symptoms (previously established as time point for a peak value in transaminases). However, for the s.c. administration route no macroscopic symptoms were seen. More specifically, no increase in transaminases was seen 11 hours post injection (1 h post the time of FcγRIIB saturation according to PK (10 h)), as shown in FIG. 15B.

[0491] With regard to clinical chemistry parameters an increase in transaminases (AST and ALT) with a peak 1 h post injection was the only parameter affected by AT-130-2 injection. These increases were just like the PLT decrease transient (FIG. 16B). The same transient increase was seen when AT-130-2 was injected i.v. and no increase in transaminases was detected when AT-130-2 was injected s.c.

[0492] A panel of cytokines including the analytes IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO, TNF-α were analyzed at different time points post injection of 200 μg i.p. Of all the analyzed cytokines IL-5, IL-6, IL-10, KC/GRO, TNF-α showed a transient increase, all peaking 1-3 hours post injection, except for IL-5 (FIG. 16C). IL-5 showed a delayed peak 3-8 hours post injection (FIG. 16C). These were the same cytokines that have been indicated to increase in some patients in the clinical studies with BI-1206.

Premedication

[0493] In order to investigate if premedication with corticosteroids could inhibit the IRRs and associated toxicities of AT-130-2, mice were premedicated with 40 mg/kg betamethasone 16-24 h and 1 h pre injection of AT-130-2. Both the IRRs and the platelet decrease seen with AT-130-2 was completely inhibited with premedication (FIG. 17A-B). Also, the increase in liver transaminases and cytokine release was less profound (FIG. 17C and data not shown). The same effect was seen when another corticosteroid, dexamethasone was assessed (data not shown).

[0494] To assess the importance of the dose of corticosteroid treatment the dose of betamethasone was decreased from 40 mg/kg to 10 mg/kg in the following experiment (FIG. 18). At 10 mg/kg both IRRs and a decrease in platelet count were seen in half of the mice indicating that a high dose of corticosteroids is needed to completely block the IRRs and associated toxicities (FIG. 18).

[0495] Further, the importance of the two doses of corticosteroid treatment was investigated by comparing the protective effect of only early (1 hours pre injection) or only late (24 hours pre injection) premedication with the two doses of corticosteroid treatment. Premedication only 1 hour pre injection could not inhibit the IRRs nor the decrease in platelet count (FIG. 19). Premedication 24 h pre injection diminished the IRRs and the platelet decrease but could not completely block these symptoms (FIG. 19), indicating that two doses of corticosteroids is needed to fully block IRRs.

[0496] Finally, the impact of antihistamine, which is a standard premedication in the clinical trials, was evaluated. Premedication with antihistamine alone did not inhibit IRRs or platelet decrease. When combining the two doses of corticosteroid treatment with antihistamine pretreatment the protective effect was retained (FIG. 20). These results were confirmed for three different types of antihistamines (Zyrlex, Zantac and Aeurius).

Conclusions

[0497] Our data demonstrates an in vivo model using intra-venous (i.v.) or intra-perinatal (i.p.) administration of anti-FcγRIIB mIgG2a surrogate (AT-130-2) in wild type mice recapitulates the tolerability profile seen with BI-1206, including IRR's, decreased platelet count, elevated transaminases (i.e. ALT and AST) and transient cytokine release. The IRR's appear 5-20 minutes after AT-130-2 injection with macroscopic symptoms including isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture and decreased blood pressure. The visual physical reaction is transient, and the animals are fully recovered 1 h after the antibody administration. The macroscopic symptoms are accompanied by a decrease in platelet count and elevated transaminases which are normalized within 8 hours. The cytokine release is acute and transient and includes IL-6, IL-5, IL-10, TNFα and KC/GRO (rodent homolog of human IL-8). The cytokine profile and kinetics is equivalent to what is seen after BI-1206 in human subjects. In the mouse model there is an apparent correlation between the IRR's and high and rapid exposure, rather than time of FcγRIIB saturation, where sub-cutaneous (s.c.) administration of AT-130-2 is better tolerated than i.p. and i.v. administration. The timing of onset of symptoms correlates with the serum concentration where receptor saturation is achieved. However, also when administrating an antibody dose that achieves receptor saturation for 6 days or longer the animals recover from all symptoms within 24 h. Sustained FcγRIIB blockade per se does not appear to be the causative of IRRs.

[0498] In this model premedication with two doses of corticosteroids (dexamethasone or betamethasone) inhibits the macroscopic IRRs as well as platelet decrease and transaminase elevation. The two doses are given s.c. 16-24 hours and i.v. 30-60 minutes prior to antibody administration. The prevention of macroscopic symptoms in the mice by corticosteroids is dose-dependent and importantly the timing of premedication is crucial. The dose 16-24 hours prior to antibody administration is imperative in order to gain the protective effect. If corticosteroids are only given 30-60 minutes prior to antibody administration no protective effect with regard to the macroscopic symptoms is seen, whereas the dose 16-24 hours prior to antibody administration alone partially improves tolerability. When both doses are given, inhibition of the macroscopic symptoms, platelet decrease, transaminase elevation, and cytokine release is achieved.

[0499] Dosing of human patients according to the corticosteroid-based regimen identified in the mouse model protected against IRRs and allowed for administration of higher doses, which are likely to be associated with stronger antitumor activity, of the studied anti-FcgRIIB antibody.

Example 4B—Other Targets

Materials & Methods

Test and Control Substances

[0500] The anti-mouse CD32b clone was transiently expressed in HEK293 cells. The specificity of the batch was demonstrated in a luminescence-based enzyme linked immunosorbent assay (ELISA) or in flow cytometry analyses. Endotoxin-levels of antibodies were found to be <0.1 IU/mL as determined by the LAL-Amoebocyte test. The anti-mouse CD40, EGFR and CSFR1 antibodies were purchased from BioXcell or Absolute Antibody (see table below) and the anti-mouse FcγRIII antibody AT154-2 was a gift from University of Southampton. Alternatively, AT154-2 as rat IgG2b isotype may be purchased from, for example, BioRad, Argio Biolaboratories (ARG23942) or LSBio (LS-C745656) which is then converted into IgG2a format using any well-known method.

TABLE-US-00014 Antibody clone Description Reference AT-130-2 Mouse anti-mouse CD32b (See comments above IgG2a Example 1) FGK4.5/FGK45 anti-mouse CD40 BP0016-2, BioXcell, Rat IgG2a 7A7 Mouse IgG2a anti-mouse EGFR Ab00134-2.0, Absolute Antibody AFS98 Rat IgG2a anti-mouse CSFR1 BE0213, BioXcell AT154-2 Mouse anti-mouse FcγRIII (See comments above IgG2a the table)

Mice

[0501] Six to eight weeks-old (17-20 g) female C57/BL6 mice were obtained from Taconic. Mice were injected intra-venous (i.v.), with 200 μg/mouse of the different antibodies.

Premedication

[0502] For the corticosteroid treatment, Betapred (betamethasone, VNR: 008938, Alfasigma S.P.A.) or Dexamethasone (Cat. No: S1322, Batch No: 02, Selleckchem) was used.

Animal Monitoring

[0503] Mice were monitored post injection with regard to changes in behavior and macroscopic symptoms such as isolation, mobility, and fur condition. Macroscopic IRRs scoring system of 0-2 was set up based on the observations:

TABLE-US-00015 Scoring Macroscopic symptoms 0 No visible symptoms 1 Isolation, decreased activity 2 Isolation, decreased activity, impaired balance, piloerection, hunching followed by un-natural body posture

Blood Sampling

[0504] Blood samples were collected from vena saphena for instant blood count analysis.

Platelet Count

[0505] Platelet counts were analyzed in fresh blood using a Vetscan (Vetscan HM5 Abaxis, Triolab).

Conclusions

[0506] This example shows that the model described herein can distinguish between antibody molecules that induce tolerability issues and those that do not. It further shows that premedication can inhibit IRRs related to different antibodies and targets.

[0507] This example also shows that antibodies that induce IRRs also induce thrombocytopenia. Furthermore, it demonstrates that premedication can inhibit thrombocytopenia related to different antibodies and targets.

EMBODIMENTS OF THE INVENTION

[0508] Certain embodiments of the invention will be described with reference to the following numbered paragraphs: [0509] 1. A therapeutic system for use in improving tolerability of an antibody molecule that specifically binds to FcγRIIb in a subject, wherein the therapeutic system comprises: [0510] (i) an antibody molecule that specifically binds to FcγRIIb, wherein the antibody molecule is administered to the subject as at least a first dose and a second dose; and [0511] (ii) a corticosteroid, wherein the first dose of the antibody molecule is lower than the maximum therapeutically effective dose of the antibody molecule; and wherein the corticosteroid is administered to the subject before the first dose of the antibody molecule. [0512] 2. A combination comprising an antibody molecule and a corticosteroid for use in a dosage regimen for improving tolerability of an antibody molecule that specifically binds to FcγRIIb in a subject, wherein the dosage regimen comprises the following steps: [0513] (i) administration of a corticosteroid before administration of a first dose of the antibody molecule; [0514] (ii) administration of the first dose of the antibody molecule that specifically binds to FcγRIIb that is lower than the maximum therapeutically effective dose; and [0515] (iii) administration of a second dose of the antibody molecule that specifically binds to FcγRIIb, wherein the first dose of the antibody molecule is administered prior to the second dose. [0516] 3. Use of: [0517] (i) an antibody molecule that specifically binds to FcγRIIb; and [0518] (ii) a corticosteroid, in the manufacture of a medicament for improving tolerability of an antibody molecule that specifically binds to FcγRIIb in a subject, wherein the medicament comprises at least a first dose and a second dose of the antibody molecule; and wherein the first dose of the antibody molecule is lower than the maximum therapeutically effective dose of the antibody molecule; and wherein the corticosteroid is administered before the first dose of the antibody molecule. [0519] 4. A method for improving tolerability of an antibody molecule that specifically binds to FcγRIIb in a subject comprising: [0520] (i) administration of a corticosteroid before administration of a first dose of the antibody molecule; [0521] (ii) administration of the first dose of the antibody molecule that specifically binds to FcγRIIb that is lower than the maximum therapeutically effective dose; and [0522] (iii) administration of a second dose of the antibody molecule that specifically binds to FcγRIIb, wherein the first dose of the antibody molecule is administered prior to the second dose. [0523] 5. The system, combination for use, use, or method of paragraphs 1-4, wherein the system, combination for use, use, or method further comprises administration of one or more therapeutic antibodies for the treatment of cancer in a subject. [0524] 6. The system, combination for use, use, or method of paragraph 5, wherein the therapeutic antibody is selected from: rituximab; pembrolizumab; nivolumab; cemiplimab; camrelizumab; dostarlimab; obinutuzumab; ofatumumab, and biosimilars or equivalents thereof. [0525] 7. The system, combination for use, use, or method of paragraphs 1-6, wherein the corticosteroid is administered to the subject at a time point from 10 minutes to 48 hours before the first dose of the antibody molecule that specifically binds to FcγRIIb. [0526] 8. The system, combination for use, use, or method of paragraph 7, wherein the corticosteroid is administered to the subject at a time point from 10 minutes to 24 hours before the first dose of the antibody molecule that specifically binds to FcγRIIb. [0527] 9. The system, combination for use, use, or method of paragraphs 1-6, wherein the corticosteroid is administered as a first dose and a second dose, and wherein the first dose of the corticosteroid is administered at a time point from 16 hours to 48 hours before the first dose of the antibody molecule that specifically binds to FcγRIIb, and wherein the second dose of the corticosteroid is administered at a time point from 10 minutes to 2 hours before the first dose of the antibody molecule that binds specifically to FcγRIIb. [0528] 10. The system, combination for use, use, or method of paragraph 9, wherein a further dose of corticosteroid is administered at a time point from 16 hours to 48 hours before the second dose of the antibody molecule that specifically binds to FcγRIIb. [0529] 11. The system, combination for use, use, or method of paragraphs 1-10, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is administered at a time point from one to 24 hours before the second dose of the antibody molecule that specifically binds to FcγRIIb. [0530] 12. The system, combination for use, use, or method of paragraphs 1-10, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is administered about one hour before the second dose of the antibody molecule that specifically binds to FcγRIIb. [0531] 13. The system, combination for use, use, or method of paragraphs 1-10, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is administered about 24 hours before the second dose of the antibody molecule that specifically binds to FcγRIIb. [0532] 14. The system, combination for use, use, or method of paragraphs 1-10, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is administered from 24 hours to 48 hours before the second dose of the antibody molecule that specifically binds to FcγRIIb. [0533] 15. The system, combination for use, use, or method of paragraphs 1-14, wherein the corticosteroid is administered at a dose of 4 mg or greater. [0534] 16. The system, combination for use, use, or method of paragraphs 1-15, wherein the corticosteroid is administered at a dose of 12 mg or greater. [0535] 17. The system, combination for use, use, or method of paragraphs 1-14, wherein the corticosteroid is administered at a dose of from 4 mg to 20 mg. [0536] 18. The system, combination for use, use, or method of paragraph 17, wherein the corticosteroid is administered at a dose of from 12 mg to 20 mg. [0537] 19. The system, combination for use, use, or method of paragraph 17, wherein the corticosteroid is administered at a dose of from 4 mg to 12 mg. [0538] 20. The system, combination for use, use, or method of paragraphs 1-19, wherein the corticosteroid is dexamethasone or betamethasone or a combination of dexamethasone and betamethasone. [0539] 21. The system, combination for use, use, or method of paragraphs 1-20, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is lower than the maximum tolerated therapeutic dose. [0540] 22. The system, combination for use, use, or method of paragraphs 1-21, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is at least 50% lower than the maximum therapeutically effective dose. [0541] 23. The system, combination for use, use, or method of paragraphs 1-22, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is administered at a dose of from 0.2 mg/kg to 0.6 mg/kg. [0542] 24. The system, combination for use, use, or method of paragraph 23, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is administered at a dose of from 0.3 mg/kg to 0.5 mg/kg. [0543] 25. The system, combination for use, use, or method of paragraphs 1-24, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is administered at a dose of from 20 mg to 40 mg. [0544] 26. The system, combination for use, use, or method of paragraph 25, wherein the first dose of the antibody molecule that specifically binds to FcγRIIb is administered at a dose of about 30 mg. [0545] 27. The system, combination for use, use, or method of paragraphs 1-26, wherein the second dose of the antibody molecule that specifically binds to FcγRIIb is a therapeutically effective dose. [0546] 28. The system, combination for use, use, or method of paragraphs 1-27, wherein the second dose of the antibody molecule that specifically binds to FcγRIIb is the maximum tolerated therapeutic dose or the maximum feasible therapeutic dose. [0547] 29. The system, combination for use, use, or method of paragraphs 1-27, wherein the second dose of the antibody molecule that specifically binds to FcγRIIb is lower than a therapeutically effective dose. [0548] 30. The system, combination for use, use, or method of paragraphs 1-29, wherein further additional doses of the antibody molecule that specifically binds to FcγRIIb are administered to the subject following the second dose of the antibody molecule that specifically binds to FcγRIIb. [0549] 31. The system, combination for use, use, or method of paragraphs 1-30, wherein infusion related reactions associated with the administration of the antibody molecule that specifically binds to FcγRIIb are reduced or eliminated. [0550] 32. The system, combination for use, use, or method of paragraphs 1-31, wherein changes to the body temperature and/or platelet count and/or blood levels of liver enzymes of the subject are reduced (and are preferably reduced to acceptable levels) for at least 24 hours following administration of the second dose of the antibody molecule that specifically binds to FcγRIIb. [0551] 33. The system, combination for use, use, or method of paragraphs 1-32, wherein the antibody molecule that specifically binds to FcγRIIb is capable of binding one or more Fcγ receptors via its Fc region. [0552] 34. The system, combination for use, use, or method of paragraphs 1-33, wherein the antibody molecule that specifically binds to FcγRIIb has a light chain sequence of SEQ ID No: 1 and a heavy chain of SEQ ID No: 2. [0553] 35. The system, combination for use, use, or method of paragraphs 1-34, wherein the first and second doses of the antibody molecule that specifically binds to FcγRIIb are formulated for intravenous delivery to the subject. [0554] 36. The system, combination for use, use, or method of paragraphs 1-35, wherein the corticosteroid is formulated for intravenous or oral delivery to the subject. [0555] 37. A kit comprising: [0556] (i) an antibody molecule that specifically binds to FcγRIIb, optionally as defined in paragraphs 33 and/or 34; [0557] (ii) a corticosteroid, optionally as defined in any one of paragraphs 15-20; and [0558] (iii) optionally, instructions for use, wherein the antibody molecule is provided as a first dose and a second dose, wherein the first dose of the antibody molecule is lower than the maximum therapeutically effective dose of the antibody molecule, further optionally wherein the first dose is as defined in paragraphs 11-14 and 21-26, further optionally wherein the second dose is as defined in paragraphs 27-29. [0559] 38. The kit of paragraph 37, wherein the kit is for improving the tolerability of the antibody molecule in a subject. [0560] 39. The kit of paragraph 37 or 38, wherein the corticosteroid is provided in a dose as defined in any one of paragraphs 12-17. [0561] 40. The kit of paragraphs 37-39, wherein the kit further comprises one or more therapeutic antibodies. [0562] 41. The kit of paragraph 40, wherein the therapeutic antibody is selected from: rituximab; pembrolizumab; nivolumab; cemiplimab; camrelizumab; dostarlimab; obinutuzumab; ofatumumab, and biosimilars or equivalents thereof. [0563] 42. The kit of paragraph 40 or 41, wherein the kit is for use in treating cancer in a subject. [0564] 43. The system, combination for use, use, method, or kit substantially as described herein with reference to the description and drawings. [0565] 44. A method for predicting if a therapeutic antibody molecule binding specifically to a human target will be associated with a tolerability issue in connection with intravenous administration to a human, comprising the following step: [0566] (i) intravenous or intraperitoneal administration of the therapeutic antibody molecule, if cross-reactive with murine target, or a surrogate antibody, to a mouse and observation of the mouse during a period following immediately after the administration of the therapeutic or surrogate antibody, wherein a display of the macroscopic symptoms isolation and decreased activity during the period followed by restoration of the state of the mouse to the normal state is an indication that the intravenous administration of the therapeutic antibody molecule to a human will be associated with a tolerability issue, and/or for predicting if a prophylactic or therapeutic treatment, an altered administration route and/or a modification of the therapeutic antibody molecule can prevent or mitigate a tolerability issue associated with intravenous administration to a human of a therapeutic antibody molecule binding specifically to a human target, comprising the following step(s) in addition to (i) as set out above: [0567] (ii) administration of a prophylactic or therapeutic agent to a mouse in conjunction with intravenous or intraperitoneal administration of the therapeutic or surrogate antibody to a mouse, and observation of the mouse during a period following immediately after the administration of the therapeutic or surrogate antibody, wherein a decreased display of the macroscopic symptoms compared to the macroscopic symptoms displayed by the mouse in (i) or no display of the macroscopic symptoms during the period is an indication that pre-treatment with the prophylactic or therapeutic agent in combination with administration of the therapeutic antibody molecule to a human can prevent or mitigate the tolerability issue that otherwise would be associated with intravenous administration of the therapeutic antibody molecule to a human; [0568] (iii) administration of the therapeutic or surrogate antibody to a mouse by a route of administration other than intravenous or intraperitoneal administration, and observation of the mouse during a period following immediately after the administration of the therapeutic or surrogate antibody, wherein a decreased display of the macroscopic symptoms compared to the macroscopic symptoms displayed by the mouse in (i) or no display of the macroscopic symptoms during the period is an indication that the other route of administration can be used for administration of the therapeutic antibody molecule to a human to prevent or mitigate the tolerability issue that would be associated with intravenous administration of the therapeutic antibody molecule to a human; and/or [0569] (iv) intravenous or intraperitoneal administration of a modified format of the therapeutic or surrogate antibody to a mouse by a route of administration other than intravenous or intraperitoneal administration, and observation of the mouse during a period following immediately after the administration of the modified therapeutic or surrogate antibody, wherein a decreased display of the macroscopic symptoms compared to the macroscopic symptoms displayed by the mouse in (i) or no display of the macroscopic symptoms during the period is an indication that administration of the therapeutic antibody molecule in the modified format to a human can be used to prevent or mitigate the tolerability issue that would be associated with intravenous administration of the therapeutic antibody molecule to a human. [0570] 45. A method according to paragraph 44, wherein a display in (i) of 1-3 additional macroscopic symptoms selected from impaired balance, piloerection, and hunching followed by un-natural body posture during the period in (i) where after the state of the mouse is restored to the normal state further strengthens the indication that the intravenous administration of the therapeutic antibody molecule to the human will be associated with a tolerability issue. [0571] 46. A method according to paragraph 44 or 45, wherein the period during which the macroscopic symptoms are displayed in (i) starts 5-10 minutes after administration of the therapeutic or surrogate antibody and ends 45-90 minutes after administration of the therapeutic or surrogate antibody, and wherein the observation period in (ii), (iii) and/or (iv) is of the same length. [0572] 47. A method according to any one of the paragraphs 44-46, wherein at least one of the following additional parameters: [0573] decreased blood pressure [0574] decreased platelet count, and or [0575] increased liver enzymes (AST/ALT). [0576] observed during the period in (i) further strengthens the indication that the intravenous administration of the therapeutic antibody molecule to a human will be associated with a tolerability issue. [0577] 48. A method for predicting if a prophylactic or therapeutic treatment can prevent or mitigate a tolerability issue associated with intravenous administration to a human of a therapeutic antibody molecule binding specifically to a human target according to any one of the claims 1-4 comprising at least steps (i) and (ii), wherein pre-treatment is used in (ii) and wherein this pre-treatment is administration of a corticosteroid to the mouse prior to injection of the therapeutic or surrogate antibody. [0578] 49. A method according to paragraph 48, wherein the pre-treatment comprises two administrations of a corticosteroid, wherein one is given 10.sup.−48 hours prior to administration of the therapeutic or surrogate antibody and the other is given 5 minutes-5 hours prior to administration of the therapeutic or surrogate antibody. [0579] 50. A method according to paragraph 49, wherein the corticosteroid is dexamethasone or betamethasone. [0580] 51. A method for predicting if an altered administration route can prevent or mitigate a tolerability issue associated with intravenous administration to a human of a therapeutic antibody molecule binding specifically to a human target according to any one of the paragraphs 44-50 comprising at least steps (i) and (iii), wherein the route of administration used in (iii) is subcutaneous administration. [0581] 52. A method for predicting if a modification of the therapeutic antibody molecule can prevent or mitigate a tolerability issue associated with intravenous administration to a human of a therapeutic antibody molecule binding specifically to a human target according to any one of the paragraphs 44-51 comprising at least steps (i) and (iv), wherein the modified format of the therapeutic or surrogate antibody used in (iv) is a modification that leads to decreased or abolished engagement of Fc receptors. [0582] 53. A method according to any one of the paragraphs 44-52, wherein the human target is selected from the group consisting of FcγRIIB, FcγRIIA and CD40. [0583] 54. A method according to paragraph 53, wherein the therapeutic antibody molecule is a human anti-FcγRIIB antibody capable of binding a human FcγR via its Fc domain and wherein the mouse surrogate antibody is an anti-FcγRIIb antibody capable of binding a mouse FcγR via its Fc domain. [0584] 55. A method according to paragraph 54, wherein the therapeutic antibody molecule is a human anti-FcγRIIB IgG1 antibody and wherein the mouse surrogate antibody is an anti-FcγRIIb mIgG2a. [0585] 56. A corticosteroid for use in a dosing regimen to prevent or mitigate a tolerability issue in connection with intravenous administration of a therapeutic antibody molecule to a subject, [0586] wherein the therapeutic antibody molecule has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of any one of the paragraphs 44-56, and/or wherein pre-treatment with the corticosteroid combination with administration of the therapeutic antibody molecule to a human has been predicted to prevent or mitigate the tolerability issue that otherwise would be associated with intravenous administration of the therapeutic antibody molecule to a human using the method of any one of the paragraphs 48-50, or any one of the paragraphs 53-55 when referring to any one of the paragraphs 48-50, [0587] and wherein the dosing regimen comprises administration of the corticosteroid to the subject in at least two doses prior to intravenous administration of the therapeutic antibody molecule, wherein one dose of the corticosteroid is administered 10-48 hours prior to start of the administration of therapeutic antibody molecule (“the first dose”) and one dose of the corticosteroid is administered 5 minutes-5 hours prior to the start of administration of the therapeutic antibody molecule (“the second dose”). [0588] 57. A corticosteroid for use in a dosing regimen to prevent or mitigate a tolerability issue in connection with intravenous administration of a therapeutic antibody molecule to a subject, [0589] wherein the therapeutic antibody molecule is an anti-FcγRIIB antibody, [0590] and wherein the dosing regimen comprises administration of the corticosteroid to the subject in at least two doses prior to intravenous administration of the therapeutic antibody molecule, wherein one dose of the corticosteroid is administered 10-48 hours prior to start of the administration of therapeutic antibody molecule (“the first dose”) and one dose of the corticosteroid is administered 5 minutes-5 hours prior to the start of administration of the therapeutic antibody molecule (“the second dose”). [0591] 58. A corticosteroid for use according to paragraph 56 or 57, wherein the first dose is given 6-36 hours prior to start of administration of the therapeutic antibody molecule and the second dose is given 15-120 minutes prior to start of administration of the therapeutic antibody molecule. [0592] 59. A corticosteroid for use according to paragraph 56-58, wherein the first dose is given 16-24 hours prior to start of administration of the therapeutic antibody molecule. [0593] 60. A corticosteroid for use according to any one of the paragraphs 56-59, wherein the second dose is given 30-60 minutes prior to start of administration of the therapeutic antibody molecule. [0594] 61. A corticosteroid for use according to any one of the paragraphs 56-60, wherein the dosing regimen comprises administration of the at least two doses of the corticosteroid prior to each infusion of the antibody during the course of antibody therapy. [0595] 62. A corticosteroid for use according to claim any one of the paragraphs 56-61, wherein the corticosteroid is dexamethasone or betamethasone or a combination of dexamethasone and betamethasone. [0596] 63. A corticosteroid for use according to any one of the paragraphs 56-62, wherein the corticosteroid is dexamethasone and wherein the first dose is 4-20 mg and the second dose is 4-25 mg. [0597] 64. A corticosteroid for use according to paragraph 63, wherein the first dose is 10-12 mg and the second dose is 20 mg. [0598] 65. A corticosteroid for use according to any one of the paragraphs 56-62, wherein the corticosteroid is betamethasone and wherein the first dose is 3.2-16 mg and the second dose is 3.2-20 mg. [0599] 66. A corticosteroid for use according to paragraph 65, wherein the first dose is 8-9.6 mg and the second dose is 16 mg. [0600] 67. A corticosteroid for use according to claim any one of the paragraphs 56-66, wherein the dosing regimen further comprises administration of an antihistamine 10 minutes-24 hours prior to start of administration of the therapeutic antibody molecule. [0601] 68. A corticosteroid for use according to any one of the paragraphs 56-67, wherein the therapeutic antibody is an Fc receptor binding antibody. [0602] 69. A corticosteroid for use according to any one of the paragraphs 56-68, wherein the therapeutic antibody is an anti-FcγRIIB antibody. [0603] 70. A corticosteroid for use according to paragraph 69, wherein the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2. [0604] 71. A therapeutic antibody molecule for use in the treatment of cancer, wherein the therapeutic antibody molecule has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of any one of the paragraphs 44-55 and/or wherein the subcutaneous route of administration of therapeutic antibody molecule to a human has been predicted to prevent or mitigate the tolerability issue that otherwise would be associated with intravenous administration of the therapeutic antibody molecule to a human using the method of paragraph 51, or any one of the paragraphs 53-55 when referring to paragraph 51, [0605] and wherein the therapeutic antibody is formulated for subcutaneous administration. [0606] 72. A therapeutic antibody molecule for use according to paragraph 71, wherein the therapeutic antibody is an anti-FcγRIIB antibody. [0607] 73. A therapeutic antibody molecule for use according to paragraph 72, wherein the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2. [0608] 74. A modified format of a therapeutic antibody molecule for use in the treatment of cancer, wherein the therapeutic antibody molecule has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of any one of the paragraphs 44-55 and/or wherein administration of the therapeutic antibody molecule in the modified format to a human has been predicted to prevent or mitigate the tolerability issue that otherwise would be associated with intravenous administration of the therapeutic antibody molecule to a human using the method of paragraph 52, or any one of the paragraph 53-55 when referring to paragraph 52, and wherein the therapeutic antibody molecule is an Fc receptor binding antibody and the modified format is an antibody having the same Fv variable sequence but having impaired or abrogated FcγR binding compared with the therapeutic antibody molecule. [0609] 75. A modified format of a therapeutic antibody molecule for use according to paragraph 74, wherein the therapeutic antibody is an anti-FcγRIIB antibody. [0610] 76. A modified format of a therapeutic antibody molecule for use according to paragraph 75, wherein the modified format of the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 295. [0611] 77. A method for preventing or mitigating a tolerability issue in connection with intravenous administration of a therapeutic antibody molecule to a subject comprising a corticosteroid dosing regimen, [0612] wherein the therapeutic antibody molecule has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of any one of the paragraphs 44-56, and/or wherein pre-treatment with the corticosteroid combination with administration of the therapeutic antibody molecule to a human has been predicted to prevent or mitigate the tolerability issue that otherwise would be associated with intravenous administration of the therapeutic antibody molecule to a human using the method of any one of the paragraphs 48-50, or any one of the paragraphs 53-56 when referring to any one of the paragraphs 48-50, [0613] and wherein the dosing regimen comprises administration of the corticosteroid to the subject in at least two doses prior to intravenous administration of the therapeutic antibody molecule, wherein one dose of the corticosteroid is administered 10-48 hours prior to start of the administration of therapeutic antibody molecule (“the first dose”) and one dose of the corticosteroid is administered 5 minutes-5 hours prior to the start of administration of the therapeutic antibody molecule (“the second dose”). [0614] 78. A method according to paragraph 77, wherein the first dose is given 6-36 hours prior to start of administration of the therapeutic antibody molecule and the second dose is given 15-120 minutes prior to start of administration of the therapeutic antibody molecule. [0615] 79. A method according to paragraph 77 or 78, wherein the first dose is given 16-24 hours prior to start of administration of the therapeutic antibody molecule. [0616] 80. A method according to any one of the paragraphs 77-79, wherein the second dose is given 30-60 minutes prior to start of administration of the therapeutic antibody molecule. [0617] 81. A method according to any one of the paragraphs 77-80, wherein the dosing regimen comprises administration of the at least two doses of the corticosteroid prior to each infusion of the antibody during the course of antibody therapy. [0618] 82. A method according to any one of the paragraphs 77-81, wherein the corticosteroid is dexamethasone or betamethasone or a combination of dexamethasone and betamethasone. [0619] 83. A method according to any one of the paragraphs 77-82, wherein the corticosteroid is dexamethasone and wherein the first dose is 4-20 mg and the second dose is 4-25 mg. [0620] 84. A method according to paragraph 83, wherein the first dose is 10-12 mg and the second dose is 20 mg. [0621] 85. A method according to any one of the paragraphs 77-82, wherein the corticosteroid is betamethasone and wherein the first dose is 3.2-16 mg and the second dose is 3.2-20 mg. [0622] 86. A method according to paragraph 85, wherein the first dose is 8-9.6 mg and the second dose is 16 mg. [0623] 87. A method according to claim any one of the paragraphs 77-86, wherein the dosing regimen further comprises administration of an antihistamine 10 minutes-24 hours prior to start of administration of the therapeutic antibody molecule. [0624] 88. A method according to any one of the paragraphs 76-86, wherein the antibody is an Fc receptor binding antibody. [0625] 89. A method according to any one of the paragraphs 77-88, wherein the antibody is an anti-FcγRIIB antibody. [0626] 90. A method according to paragraph 89, wherein the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2. [0627] 91. A method for treatment of cancer comprising subcutaneous administration of a therapeutically active amount of a therapeutic antibody molecule which has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of any one of the paragraphs 44-56 and/or wherein the subcutaneous route of administration of therapeutic antibody molecule to a human has been predicted to prevent or mitigate the tolerability issue that otherwise would be associated with intravenous administration of the therapeutic antibody molecule to a human using the method of paragraph 51, or any one of the paragraphs 53-56 when referring to paragraph 51. [0628] 92. A method according to paragraph 91, wherein the antibody is an anti-FcγRIIB antibody. [0629] 93. A method according to paragraph 92, wherein the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2. [0630] 94. A method for treatment of cancer comprising administration of a therapeutically active amount of a modified format of a therapeutic antibody, wherein the therapeutic antibody molecule has been predicted to be associated with a tolerability issue in connection with intravenous administration to a human using the method of any one of the paragraphs 44-56 and/or wherein administration of the therapeutic antibody molecule in the modified format to a human has been predicted to prevent or mitigate the tolerability issue that otherwise would be associated with intravenous administration of the therapeutic antibody molecule to a human using the method of paragraph 52, or any one of the paragraphs 53-56 when referring to paragraph 52, [0631] and wherein the therapeutic antibody molecule is an Fc receptor binding antibody and the modified format is an antibody having the same Fv variable sequence but having impaired or abrogated FcγR binding compared with the therapeutic antibody molecule. [0632] 95. A method according to paragraph 94, wherein the antibody is an anti-FcγRIIB antibody. [0633] 96. A method according to paragraph 95, wherein the anti-FcγRIIB antibody is the antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2 with a N297Q mutation in the heavy chain. [0634] 97. A therapeutic antibody molecule for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, an immunological disease, and/or an infectious disease, wherein the therapeutic antibody molecule is an anti-FcγRIIB antibody, and wherein the therapeutic antibody molecule is formulated for subcutaneous administration. [0635] 98. Use of a therapeutic antibody molecule in the manufacture of a medicament for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, an immunological disease, and/or an infectious disease, wherein the therapeutic antibody molecule is an anti-FcγRIIB antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2, and wherein the medicament is formulated for subcutaneous administration. [0636] 99. A pharmaceutical formulation comprising a therapeutic antibody molecule, wherein the therapeutic antibody molecule is an anti-FcγRIIB antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2, and wherein the pharmaceutical formulation comprises a pharmaceutically acceptable diluent or excipient, and is formulated for subcutaneous administration. [0637] 100. A therapeutic antibody molecule for use according to paragraph 97, use of a therapeutic antibody molecule according to paragraph 98, or a pharmaceutical formulation according to paragraph 99, wherein the therapeutic antibody is an Fc receptor binding antibody. [0638] 101. A therapeutic antibody molecule for use according to paragraph 97 or 100, use of a therapeutic antibody molecule according to paragraph 98 or 100, or a pharmaceutical formulation according to paragraph 99 or 100, wherein the therapeutic antibody is an anti-FcγRIIB antibody. [0639] 102. A therapeutic antibody molecule for use according to paragraph 101, use of a therapeutic antibody molecule according to paragraph 101, or a pharmaceutical formulation according to paragraph 101, wherein the therapeutic antibody has a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2. [0640] 103. A therapeutic antibody molecule for use according to paragraph 101 or 102, use of a therapeutic antibody molecule according to paragraph 101 or 102, or a pharmaceutical formulation according to paragraph 101 or 102, for treatment of cancer. [0641] 104. The pharmaceutical formulation according to any one of the paragraphs 99-103, wherein the therapeutic antibody is present at a concentration of between about 90 mg/mL and about 220 mg/mL. [0642] 105. The pharmaceutical formulation according to any one of the paragraphs 99-104, further comprising between about 5 mM and about 20 mM acetate, and/or between about 50 mM and about 250 mM NaCl, and/or about 0.05% Polysorbate 20, and/or wherein the pharmaceutical formulation is at a pH of between about pH 5.0 and about pH 5.8. [0643] 106. The pharmaceutical formulation according to any one of the paragraphs 99-105, wherein the formulation comprises: [0644] the therapeutic antibody at a concentration of 150 mg/mL; [0645] 5-5 mM acetate; [0646] 110 mM NaCl; [0647] 0.05% (w/v) Polysorbate 20; and [0648] wherein the formulation is at a pH 5.8. [0649] 107. A method for the treatment of cancer, an autoimmune disease, an inflammatory disease, an immunological disease, and/or an infectious disease in a subject, the method comprising the step of administering to the subject a therapeutic antibody molecule, wherein the therapeutic antibody molecule is an Fc receptor binding antibody, and wherein the therapeutic antibody molecule is formulated for subcutaneous administration. [0650] 108. The method of paragraph 107, wherein the Fc receptor binding antibody is an anti-FcγRIIB antibody. [0651] 109. The method of paragraph 107 or 108, wherein the Fc receptor binding antibody is an anti-FcγRIIB antibody having a light chain with SEQ ID No: 1 and a heavy chain with SEQ ID No: 2. [0652] 110. A method for the treatment of cancer, an autoimmune disease, an inflammatory disease, an immunological disease, and/or an infectious disease in a subject, the method comprising the step of subcutaneously administering to the subject a pharmaceutical formulation as defined in any one of the paragraphs 99-106. [0653] 111. The method of paragraph 109 or 110 for the treatment of cancer.