CONDITIONING REGIMEN FOR CELL TRANSPLANT

Field of the Invention

The present invention relates to a conditioning regimen for the transplant of a cell, optionally hematopoietic stem / progenitor cells (HSPC), to a subject. The invention also relates to methods for the prevention or treatment of a disease or condition in a subject by administration of a cell transplant, wherein said administration comprises the conditioning regimen of the invention.

Background of the Invention

The complex immunology involved in the transplant of cells, such as HSPC or CAR- T cells, can be problematic, particularly if there is sensitization to donor antigens prior to transplantation. The presence of donor and recipient immune systems can lead to acute and chronic rejection with both humeral and cellular components. Vigorous host versus graft reactions (HVG) and graft versus host disease (GVHD) are both observed. Often, the transplanted cells fail to successfully engraft in the recipient. Current methods seek to address these problems by pre- and post-transplant immunosuppression. Steps carried out pre-transplant may be referred to as a conditioning regimen and may include treatments that are not solely immunosuppressive. For example, radiation may be used to deplete some or all of the existing bone marrow cells in the recipient, creating space for engraftment of the transplanted cells. However, lymphocyte depleting agents are often used. Using high doses of these agents leads to a desired reduction of GvHD, but at the cost of delayed immune reconstitution or even increased host engraftment failure (because the agents also act on the transplanted cells), thereby enhancing the risk of severe infectious complications and reducing overall survival (OS). There is a need for improved conditioning regimens for the transplant of cells.

Summary of the Invention

Conditioning regimens for the transplant of cells, such as HSPC or CAR-T cells, typically include a step of reducing the numbers and/or down-modulating the activity of lymphocytes (preferably T lymphocytes) in the subject prior to transplant. This may be referred to as lymphocyte depletion, or lymphodepletion. It may be achieved by administration of an agent comprising IgG-based molecules. Preferred examples include rabbit antithymocyte globulin (rATG)(e.g., ATG-G (Grafalon) or Thymoglobulin) or an anti- CD52 monoclonal antibody (e.g. alemtuzumab, CAMPATH/Lemtrada). When applied to recipients of hematopoietic stem cells before or during conditioning, such drugs can efficiently eliminate host lymphocytes (T lymphocytes), thus preventing graft rejection. Whilst a reduction in lymphocyte numbers and/or activity may be beneficial in terms of improved cell engraftment, it of course also has negative effects, in that the agents responsible for lymphodepletion may also attack the transplanted cells, and the patient will have a less effective immune system for a prolonged time until the effects are reversed, e.g. by recovery of lymphocyte numbers. This can be a particular problem with T cell-depleting antibodies (TCD-Ab) such as rATG or anti-CD52 antibodies (such as alemtuzumab) as these generally have a long half-life and can thus continue to deplete T lymphocytes long after the transplant has been administered. A similar problem occurs with checkpoint inhibitors such as PD-1 and PD-L1 antibodies, which can also have an inhibiting effect on transplanted cells after HSCT or treatment with CAR cells.

Conditioning regimens are also used for patients with hematopoietic cancers. Such patients are not only treated with IgG-based biologies like rituximab, alemtuzumab or rATG during first and second line treatments but also during bridging therapies (Bhaskar-2021) for hematopoietic stem cells transplantation (HSCT) or to debulk the tumor burden prior to CAR T cell therapy infusions. Lowering the tumor burden through preconditioning can reduce the risk for CAR T therapy-induced tumor lysis syndrome. Furthermore, the use of biologies and other lymphodepleting measures reduces the killing of infused CAR T cells by the host T-cells, which might be a problem especially after repeated dosing with allogeneic CAR T cells. An additional reason to use lymphodepleting agents can be to open a niche for the expansion of the CAR T cells. Lymphodepleting conditioning can therefore even be beneficial before the first infusion of autologous CAR T cells.

Despite the benefits of lymphodepletion there are also drawbacks when combined with CAR T-cell treatments. CD52 positive CAR-T cells, for example, are vulnerable for depletion by remaining anti-CD52 antibodies like alemtuzumab. The inactivation or deletion of CAR-T cells may also occur using biologies like anti-CD38 and rATG such as Grafalon or Thymoglobulin, which bind to naturally expressed cell surface antigens on CAR T-cells. Furthermore, previously infused disease-related therapeutic IgGs (e.g. rituximab, cetuximab) could trigger genetically introduced antigens like the rituximab-binding RQR8 safety switches on CAR T-cells. It might be desirable to shorten the time gap between the last rituximab injection and CAR T-cell infusion, allowing for a smoother transition from e.g. debulking CD20 positive tumor cells with RTX with an effective CAR treatment. The positive and negative aspects of reducing the numbers and/or down-modulating the activity of lymphocytes therefore require careful balance. For example, conditioning regimens may use a lower intensity lymphocyte depletion (e.g. lower / less frequent doses of cell depleting agent) so that immune recovery is faster after transplant is complete, but this may result in less effective transplant and/or a slower transplant procedure overall if a longer delay is required between initiation of lymphocyte depletion and the transplant.

Alternatively, conditioning regimens may use higher intensity lymphocyte depletion (e.g. higher / more frequent doses of cell depleting agent), but this may result in a longer time to immune recovery in the patient, and the depleting agent may remain in sufficient quantities to attack the transplanted cells.

The inventors have surprisingly shown that a conditioning regimen including enzymatic inactivation of serum IgG in a subject results in significant benefits by making this balance easier to achieve, when used to rapidly inactivate an IgG-based agent to reduce the numbers and/or down-modulate the activity of lymphocytes after it has had its positive effects, thereby reducing the time in which it may exert negative effects.

The present invention provides a conditioning regimen for the transplant of a cell to a subject, comprising: a. administering to the subject an agent comprising IgG molecules, which agent reduces the numbers and/or down-modulates the activity of lymphocytes in the subject; b. subsequently administering to the subject an enzyme which inactivates serum IgG molecules in the subject; and c. subsequently administering the transplant to the subject.

The present invention also provides a method for the prevention or treatment of immune rejection of a cell transplant, the method comprising administering a cell transplant to a patient in accordance with the conditioning regimen of the invention.

The present invention also provides a method for the prevention or treatment of a disease or condition that is prevented or treated by cell transplant, the method comprising administering a cell transplant to a patient in accordance with the conditioning regime of the invention. The disease or condition may be selected from Hematological malignancies; Solid tumor cancers; Hematologic diseases; Anemias; Myeloproliferative disorders; Metabolic disorders; Environmentally-induced diseases; Viral diseases; Autoimmune diseases; Lysosomal storage disorders; and Immunodefi ci enci es . The invention also provides a conditioning regimen for the transplant of a cell to a subject, wherein the subject has previously been administered with an agent comprising IgG molecules, which agent reduces the numbers and/or down-modulates the activity of lymphocytes in the subject; wherein the conditioning regimen comprises a step of administering to the subject an enzyme which inactivates serum IgG molecules in the subject.

The present invention also provides a method for the treatment of alemtuzumab overdose, comprising administering to a patient in need thereof a therapeutically effective amount of an enzyme which inactivates serum IgG molecules in the subject.

Brief Description of the Figures

Fig 1. Mean imlifidase concentration vs. nominal time from dosing (N=15). Data BLQ are included in mean calculation as BLQ/2. SD indicated with bars.

Fig 2. In vitro cleavage of rATG by imlifidase over time. Columns indicate number of subjects with visible intact rATG (Thymoglobulin) on Western blot post-imlifidase (N=l 1).

Fig.3. Imlifidase cleaves rabbit and human but not horse IgG-based therapeutic antibodies. IgG-based biologies have been incubated with a titration of imlifidase supplemented with BSA as carrier protein. Samples were incubated for 90 min at 37°C before being separated on SDS-PAGE (4-20% gradient). Polyclonal rabbit IgG mixtures at Img/mL (panels A-Thymoglobulin and B-Grafalon), and the human IgGl -based anti-CD52 mAb alemtuzumab at 1 mg/mL (panel C), are easily cleaved into single- and fully cleaved IgG by imlifidase. Equine ATGAM (Img/mL) (panel D) is largely resistant to imlifidase cleavage, only a small fraction can be detected to be cleaved, as evidenced by the faint formation of Fc/2 when incubated with imlifidase.

Fig.4. Imlifidase has an effect on NK cell survival after rATG and alemtuzumab challenge. Cytotoxicity of NK cells against K562 cells and the role of rATG and alemtuzumab measured with cytotoxicity calcein release assay. Cytotoxic activity against casein-labeled K562 target cells was measured as a readout for survival and functionality of eNK cells after preincubation with intact rATG (Grafalon) (A) or intact alemtuzumab (D). Both reagents impact the capacity of eNK cells to efficiently lyse the K562 target cells. rATG shows a dose-dependent cytotoxicity in the tested range of 2 to 250 pg/mL. After incubation of eNK cells with 100 and 250 pg/mL rATG the least lysis (%) of the K562 target cells can be observed (A). Lysis of K562 target cells by eNK cells is already prevented at 2 pg/mL alemtuzumab (D). Challenging the eNK cells with rATG F(ab‘)2 preparations or with equimolar concentrations of the intact rATG protects the eNK effector cells, allowing only for an partially increased lysis of K562 target cells (B) even at 73,3 and 183,2 pg/mL F(ab‘)2. In contrast, alemtuzumab F(ab‘)2 fragments hardly have any cytotoxic effect on eNK cells, even allowing them to handle concentrations of alemtuzumab-F(ab‘)2 as high as 75,3 pg/mL (E). Imlifidase-generated 2*Fc/2 from rATG (C) and alemtuzumab 2*Fc/2 (F) do not seem to have a cytotoxic effect on eNK cells.

Fig. 5 ADCP effector function of alemtuzumab is prevented by imlifidase. Alemtuzumab was pre-treated with different concentrations of imlifidase and separated on SDS PAGE (4- 20%) to identify samples with intact IgG (well 14), F(ab’)2 (well 1) and scIgG (well 9) fragments (A). Calcein-labeled NuDULl target cells were opsonized with 30 pg/mL of the respective alemtuzumab preparations before incubation with FarRed labeled THP1 effector cells. After 90 min at 37°C, the cells were analyzed by flow cytometry to identify double positive, i.e. phagocytosed cells. At a concentration of 30 pg/mL, scIgG has the same ability to induce phagocytosis as intact alemtuzumab. Fully cleaved F(ab’)2 alemtuzumab does not opsonize the NuDULl cells for phagocytosis by THP1 cells (B). scIgG- and intact- alemtuzumab were further compared using different concentrations for opsonization. scIgG alemtuzumab loses its opsonization effect down to background levels at around 2 pg/mL while intact alemtuzumab still has the same activity as seen at 30 pg/mL (C).

Fig. 6 CD3+ cells are depleted in whole blood in vitro by alemtuzumab and rATG (Thymoglobulin) in dose dependent manner. Peripheral blood was collected from a healthy volunteer in sodium heparin tubes. The whole blood was incubated with titrations of alemtuzumab (A) and Thymoglobulin (Genzyme) (B) for 4 and 24 hours. After RBC lysis the cells were stained with anti-CD3-PE antibody and analyzed using a Accuri C6 flow cytometer.

Fig. 7 Alemtuzumab-induced CD3+ T-cell depletion in whole blood can be prevented with imlifidase. Peripheral blood was collected from a healthy volunteer in sodium heparin tubes and pre-treated with indicated amounts of imlifidase. After 6 hours alemtuzumab (100 pg/mL) was added and incubated for another 16 hours. An aliquot of the samples was removed for plasma preparation. The plasma was analyzed running an SDS-PAGE to follow the IgG cleavage in the blood (A). RBC were lysed from the remaining samples and the PBMC stained with CD3-PE for flow cytometry using an Accuri C6 to detect the T cells. The data shows that alemtuzumab can efficiently deplete the CD3 -positive cell population in whole blood and that a concentration of approx. 4 pg/mL imlifidase is sufficient to cleave all serum IgG and 100 pg/mL alemtuzumab (B). Fig. 8. Imlifidase treatment does not protect CD3+ T-cells from depletion by ATGAM.

Whole blood from a healthy donor was treated in vitro for 6 hours with indicated concentrations of imlifidase before adding 500 pg/mL ATGAM or horse IgG (no ATGAM) as control for 16 hours. Plasma aliquots were prepared for SDS-PAGE analysis. Precision Plus MW standard (5 pL) was used as control (A). The remaining samples were lysed for RBC and labelled with PE-conjugated anti-CD3 antibody and analyzed on Accuri C6 flow cytometer (B).

Fig. 9. Cytotoxic effect of alemtuzumab can be alleviated by complement inhibition with eculizumab. Whole blood of a healthy donor was pre-treated with or without the anti-C5 eculizumab (200 pg/mL) and incubated at RT for 30 mins. The samples were treated with either 30 pg/mL intact IgG alemtuzumab, imlifidase-generated F(ab’)2, or scIgG alemtuzumab. In addition intact equine IgG ATGAM (200 pg/mL) was also incubated in the presence or absence of the complement inhibitor eculizumab for 4 hours at 37°C, 5% CO2. The blood samples were RBC-lysed, centrifuged, resuspended, and stained with a PE- conjugated anti-CD3 before analysis by flow cytometer using an Accuri C6.

Fig. 10. Clearance of in vivo generated F(ab’)2 fragments.

Fifteen healthy individuals from a Phase I Study (study number 18-HMedIdeS-15) received one i.v. dose of imlifidase (0,25 mg/kg body weight). Predose samples and post-dose up to 144 hours were collected and analyzed using a 10 well 4-20% Mini-PROTEAN®TGX™ Stain Free gel, exemplified for ID-2 (A). Five pL molecular size standard (PPP) were used. Samples were separated at 200 V for 40 min. The gels were activated and scanned using ChemiDoc Imaging system. The gel images from 15 individuals were evaluated using a visual scoring the presence (+) or absence (-) of the F(ab’)2 fragments over time (B).

Fig. 11. Imlifidase treatment reduces phagocytosis of Grafalon and Thymoglobulin opsonized platelets by THP-1 cells. Calcein stained Pure-Platelet Rich Plasma (P-PRP) cells from five donors were opsonized with and without imlifidase-treated Grafalon (100 pg /ml) or Thymoglobulin (200 pg /ml) before adding phagocytic FarRed labeled THP1 effector cells. After 90 min incubation the cells were analysed by flow cytometry for Calcein and FarRed expression as shown in dot plot (Fig. I la). All Calcein positive populations were further divided into “Calcein + / FarRed-” (platelets only) and “Calcein + / FarRed +” double positive cells (phagocytosed platelets) as shown (Fig.1 lb). The calculated percentage of the Calcein/FarRed double positive is based on all Calcein positive cells set as 100%. Changes in phagocytosis with and without imlifidase treatment is shown for Grafalon (Fig.l 1c) and Thymoglobulin (Fig.1 Id) for five individuals. Imlifidase treatment of both rabbit ATGs leads to significant reduction in phagocytosis (paired T test analysis, p < 0.05).

Fig. 12

Calcein release assays were performed to test NK cell-mediated cytotoxicity, preceded by two preincubation periods (preincubation I and II).

Fig. 13

Flow cytometric results of eNKs of one representative NK cell donor.

Fig. 14

Influence of ATG-G (Grafalon) with or without pre-cleavage by imlifidase on NK cell- mediated lysis of K562 cells based on calcein release detection. SD denotes means of triplicates, (a) 4 individual donors. First experiment, (b) 3 individual donors. Second experiment. Grafalon is referred to as “ATG” in the figure.

Fig. 15

Influence of alemtuzumab with or without pre-cleavage by imlifidase on NK cell-mediated lysis of K562 cells based on calcein release detection. SD denotes means of triplicates. 4 individual donors, (a) First experiment, (b) Second experiment.

Fig. 16

Influence of Thymoglobulin with or without pre-cleavage by imlifidase on NK cell-mediated lysis of K562 cells based on calcein release detection. SD denotes means of triplicates. 4 individual donors, (a) First experiment, (b) Second experiment.

Fig. 17

Influence of ATG-G (Grafalon) with or without pre-cleavage by imlifidase on T cell viability. Annexin V/ eFlour 780 staining of freshly isolated PBMCs from 4 different donors preincubated with ATG (Grafalon) or rabbit control AB (rlgG) +/- pre-cleavage by imlifidase. Live, early apoptotic, late apoptotic and necrotic portions (%) of CD3+ T cells are shown as means (%) + SD. (a) First experiment, (b) Second experiment.

Influence of ATG (Grafalon) with or without pre-cleavage by imlifidase on NK cell viability. Annexin V/ eFlour 780 staining of freshly isolated PBMCs from 4 different donors preincubated with ATG (Grafalon) or rabbit control AB (rlgG) +/- pre-cleavage by imlifidase. Live, early apoptotic, late apoptotic and necrotic portions (%) of CD56+ NK cells are shown as means (%) + SD. (c) First experiment, (d) Second experiment.

Grafalon is referred to as “ATG” in the figure. Fig. 18

Influence of alemtuzumab with or without pre-cleavage by imlifidase on T cell viability. Annexin V/ eFlour 780 staining of freshly isolated PBMCs from 4 different donors preincubated with alemtuzumab or human isotype-matched control AB (hlgGl) +/- precleavage by imlifidase. Live, early apoptotic, late apoptotic and necrotic portions (%) of CD3+ T cells are shown as means (%) + SD. (a) First experiment, (b) Second experiment. Influence of alemtuzumab with or without pre-cleavage by imlifidase on NK cell viability. Annexin V/ eFlour 780 staining of freshly isolated PBMCs from 4 different donors preincubated with alemtuzumab or human isotype-matched control AB (hlgGl) +/- pre- cleavage by imlifidase. Live, early apoptotic, late apoptotic and necrotic portions (%) of CD3+ T cells are shown as means (%) + SD. (c) First experiment, (d) Second experiment. Fig. 19

Influence of Thymoglobulin with or without pre-cleavage by imlifidase on T cell viability. Annexin V/ eFlour 780 staining of freshly isolated PBMCs from 4 different donors preincubated with Thymoglobulin or rabbit control AB (rlgG) +/- pre-cleavage by imlifidase. Live, early apoptotic, late apoptotic and necrotic portions (%) of CD3+ T cells are shown as means (%) + SD. (a) First experiment, (b) Second experiment.

Influence of Thymoglobulin with or without pre-cleavage by imlifidase on NK cell viability. Annexin V/ eFlour 780 staining of freshly isolated PBMCs from 4 different donors preincubated with Thymoglobulin or rabbit control AB (rlgG) +/- pre-cleavage by imlifidase. Live, early apoptotic, late apoptotic and necrotic portions (%) of CD3+ T cells are shown as means (%) + SD. (c) First experiment, (d) Second experiment.

Fig. 20: Imlifidase protects CAR-J T-cells from preconditioning treatment.

PBMCs were incubated with the preconditioning treatment antibodies Grafalon (rATG, 20 pg/mL) or Thymoglobulin (Thymo) (40 pg/mL), as well as PBS or unspecific rabbit IgG (r- IgG) (40 pg/mL) as controls. After 2 h incubation in the presence of complement, imlifidase (15 pg/mL) or PBS were added for 60 min as indicated prior to the addition of CFSE-labeled CAR-J T-cells and incubated for an additional 2 h. All cells were subsequently stained for CD3 and 7AAD and analyzed by flow cytometry to be quantified for dead cells (% 7AAD positive), (a) PBMC T-cells (CD3+/CFSE-) treated with Grafalon, Thymoglobulin, or negative control were analyzed for % 7AAD positive cells, (b) Either 0 or 15 pg/mL imlifidase were added to the preconditioning-treated PBMCs prior to the addition of CFSE- labeled CAR-J cells. The impact of imlifidase on CAR-J T-cell (CD3+/CFSE+) survival was analyzed in the 7AAD+ population. Error bars represent standard deviation of duplicates. Fig. 21: Treatment schematic

Brief Description of the Sequences

SEQ ID NO: 1 is the full sequence of IdeS including N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP_010922160.1

SEQ ID NO: 2 is the mature sequence of IdeS, lacking the N terminal methionine and signal sequence. It is also available as Genbank accession no. ADF13949.1

SEQ ID NO: 3 is the full sequence of IdeZ including N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP 014622780.1.

SEQ ID NO: 4 is the mature sequence of IdeZ, lacking the N terminal methionine and signal sequence.

SEQ ID NO: 5 is the sequence of a hybrid IdeS/Z. The N terminus is based on IdeZ lacking the N terminal methionine and signal sequence.

SEQ ID NOs: 6 to 25 are the sequences of exemplary proteases for use in the methods of the invention.

SEQ ID NO: 26 is the sequence of an IdeS polypeptide. Comprises the sequence of SEQ ID NO: 2 with an additional N terminal methionine and a histidine tag (referred to herein as pCART124).

SEQ ID NO: 27 is the sequence of an IdeZ polypeptide. Comprises the sequence of SEQ ID NO: 4 with an additional N terminal methionine and a histidine tag (referred to herein as pCART144).

SEQ ID NO: 28 is the sequence of an IdeS/Z polypeptide. Comprises the sequence of SEQ ID NO: 5 with an additional N terminal methionine and a histidine tag (referred to herein as pCART145).

SEQ ID NO: 29 is the contiguous sequence PLTPEQFRYNN, which corresponds to positions 63-73 of SEQ ID NO: 3.

SEQ ID NO: 30 is the contiguous sequence PPANFTQG, which corresponds to positions 58- 65 of SEQ ID NO: 1.

SEQ ID NO: 31 is the contiguous sequence DDYQRNATEAYAKEVPHQIT, which corresponds to positions 35-54 of SEQ ID NO: 3.

SEQ ID NO: 32 is the contiguous sequence DSFSANQEIRYSEVTPYHVT, which corresponds to positions 30-49 of SEQ ID NO: 1.

SEQ ID NOs: 33 to 55 are nucleotide sequences encoding proteases set out above. SEQ ID NOs: 56 to 69 are the sequences of exemplary proteases for use in the methods of the invention.

SEQ ID NO: 70 is the contiguous sequence NQTN, which corresponds to positions 336-339 of SEQ ID NO: 1.

SEQ ID NO: 71 is the contiguous sequence DSFSANQEIR YSEVTPYHVT, which corresponds to positions 30-49 of SEQ ID NO: 1.

SEQ ID NOs: 72 to 86 are nucleotide sequences encoding polypeptides disclosed herein.

SEQ ID NO: 87 is the sequence SFSANQEIRY SEVTPYHVT, which corresponds to positions 31-49 of SEQ ID NO: 1.

SEQ ID NO: 88 is the sequence DYQRNATEAY AKEVPHQIT, which corresponds to positions 36-54 of the IdeZ polypeptide NCBI Reference Sequence no WP 014622780.1. SEQ ID NO: 89 is the sequence DDYQRNATEA YAKEVPHQIT, which may be present at the N terminus of a polypeptide of the invention.

SEQ ID NO: 90 is the mature sequence of EndoS (Endoglycosidase of S. pyogenes).

SEQ ID NO: 91 and SEQ ID NO: 92 are further exemplary proteases for use in the methods of the invention. SEQ ID NO: 92 is the same as SEQ ID NO: 91, except it lacks the first twenty residues at the N terminus of SEQ ID NO: 1 consisting of the contiguous sequence DDYQRNATEA Y AKEVPHQIT.

Detailed Description of the Invention

General

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes “polypeptides”, and the like.

A “polypeptide” is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term “polypeptide” thus includes short peptide sequences and also longer polypeptides and proteins. As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including both D or L optical isomers, and amino acid analogs and peptidomimetics.

The terms “patient” and “subject” are used interchangeably and typically refer to a human. References to IgG typically refer to human IgG unless otherwise stated.

In some embodiments, the term “substantially” means that an agent can reduce, down-modulate or inactivate by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% compared to a control in the absence of said agent.

Amino acid identity as discussed above may be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Set. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Set. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Alternatively, the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, 387-395).

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Conditioning regimen — lymphocyte depletion or down-modulation

The present invention provides a conditioning regimen for the transplant of a cell to a subject, comprising: a. administering to the subject an agent comprising IgG molecules, which agent reduces the numbers and/or down-modulates the activity of lymphocytes in the subject; b. subsequently administering to the subject an enzyme which inactivates serum IgG molecules in the subject; and c. subsequently administering the transplant to the subject.

Step (a) may be conducted by any suitable method and using any suitable agent. The same agent or combination of agents may be effective to reduce the numbers and/or down- modulate the activity of more than one type of lymphocyte. This may be referred to as lymphocyte depletion or lymphodepletion. The agents may be referred to as lymphodepleting agents. The agents are preferably administered at a dose sufficient to substantially reduce the numbers and/or substantially down-modulate the activity of lymphocytes in the subject. Preferred agents target T lymphocytes and/or B lymphocytes, with T lymphocytes most preferred. The agent may also deplete NK cells and/or non- malignant hematopoietic disease precursors.

Exemplary agents suitable for the depletion of lymphocytes (which may be referred to as lymphodepleting agents) are known in the art and include:

- An anti-CD52 antibody, such as alemtuzumab (also known as Campath-Hl). Alemtuzumab is a humanized monoclonal antibody against CD52 and causes depletion of T and B lymphocytes, NK cells, monocytes, granulocytes, and dendritic cells, which all express CD52. Three mechanisms contribute to the depletion of these cells by alemtuzumab: CDC, ADCC and apoptosis. The half-life of alemtuzumab is generally reported at 12 days (288h), but in practice it depends on the concentration of its target. The plasma half-life of alemtuzumab is shorter (around 6 days) in patients with CLL with a large tumor burden due to cytotoxicity of malignant cells and clearance from the blood. In contrast, after successful treatment of a CLL patients, when CD52 levels decrease, the half-life of alemtuzumab increases to 8-21 days. Maintenance of a therapeutic concentration of 100 ng/ml for 56 days after HSPC transplant has been reported. No antidote to accidental overdose of alemtuzumab is known and it is unlikely that alemtuzumab would be removed with hemodialysis due to its size (150 kDa). As such there is currently no effective way to reduce the concentration of alemtuzumab in a patient (such as HSPC transplant recipient) to reduce the risk of relapse and infection. An enzyme as disclosed herein which inactivates serum IgG molecules can be used to achieve this goal, in particular an IgG cysteine protease enzyme as discussed below, preferably imlifidase. Thus, also disclosed herein is the treatment of alemtuzumab overdose by administration of such an enzyme, preferably imlifidase.

- A polyclonal mixture of non-human antibodies directed against human lymphocyte antigens, typically human T and/or B lymphocyte antigens. Various mixtures are well known in the art, and may commonly be referred to by the general term ATG, and specifically rATG (for rabbit originating mixtures) or hATG (for horse originating mixtures). The term “ATG” is taken from the International Non-Proprietary Names for such mixtures, which include (rabbit) anti-human T-lymphocyte immunoglobulin, or (rabbit) anti-human thymocyte globulin. Different “ATG” mixtures will have different antibody profiles and so may have differences in their precise immunosuppressive activities. However, all are a mixture of antibodies intended to bind to various T and B cell antigens, leading to T- and B lymphocyte depletion via (i) apoptosis via activation-induced cell death, (ii) antibody-dependent cell- mediated cytotoxicity, and (iii) complement-dependent cytotoxicity. Currently there are two ATG preparations available for use as lymphodepleting agents in human patients. Both are prepared in rabbits and so may be interchangeably referred to herein by the general term rATG, unless a specific mixture is intended, in which case this will be specified. The two mixtures differ in terms of the immunogen used to raise them and as a consequence each has different antigen specificities. The first mixture may be referred to as ATG-F (for Fresenius) or ATG-G (for Grafalon) and is produced by immunizing rabbits with the T-cell leukemia line Jurkat. The antibody profile in this mixture is rarely against CD3, CD4, CD44, and HLA-DR but targeting CD28, CD29, CD45, CD49, CD98, CD147, and more antibodies directed against CD 107 (an antigen expressed on T cells during degranulation following antigenic stimulation). It has a reported half-life of 2-3 days but can be measured in the recipients’ blood for up to 5 weeks after infusion. The second mixture may be referred to as Thymoglobulin and is produced by immunizing rabbits with fresh human thymocytes. The antibody profile in this mixture is typically against CD2, CD3, CD4, CD8, CD1 la, CD18, CD25, CD44, CD45, HLA.DR, HLA Class I heavy chains and b2-microglobulin. It has a reported half-life of 30 days, with a total dose of 10 mg/kg for 4 days (at Days -4 to -

1) detectable for 8 weeks after infusion and active agent still detectable at Day +28.

- Other agents may include any one or more of:

Other agents primarily depleting T cells

- a panel of antibodies including anti-CD4, anti-CD8, and anti-CD90; or

- an anti-CD117 antibody; or

- an anti-CD47 antibody; or

- an anti-CD45 antibody; or

- an anti-CD38 antibody (for example daratumumab); or

- abatacept; or

- an immunotoxin targeting T cells; or

Other agents primarily depleting B cells (optionally including plasma cells)

- an anti-CD20 antibody (such as rituximab);

- an anti-CD19 antibody;

- an immunotoxin targeting B cells, such as an anti-CD20 immunotoxin (for example MT-3724).

The conditioning regimen may additionally include administration also of a non-IgG based agent that is suitable to reduce the numbers and/or down-modulates the activity of lymphocytes in the subject. For example, any of busulfan, cyclophosphamide, fludarabine, treosulfan, cyclosporine, and tacrolimus may be used to deplete or inactive T cells; and any of bortezomib, fludarabine, and cyclophosphamide may be used to deplete or inactive B cells. Other agents primarily blocking costimulating factors: an anti-PD-1 antibody an anti-PD-Ll antibody

Conditioning regimen - enzyme

The present invention provides a conditioning regimen for the transplant of a cell to a subject, comprising: a. administering to the subject an agent comprising IgG molecules, which agent reduces the numbers and/or down-modulates the activity of lymphocytes in the subject; b. subsequently administering to the subject an enzyme which inactivates serum IgG molecules in the subject; and c. subsequently administering the transplant to the subject.

The amount of said enzyme administered in step (b) is preferably sufficient to inactivate all or substantially all IgG molecules present in the serum of the subject. If necessary, more than one IgG-inactivating enzyme can be administered in combination, including simultaneously or sequentially, in any order.

The term “serum IgG molecule(s)” or “IgG molecule(s) present in the serum” refers to any gamma immunoglobulin (IgGl, IgG2, IgG3 and IgG4) molecule which is present in human tissue or in circulation prior to a method of the invention being carried out. Such IgG molecules may have been produced endogenously from an individual’s B-cells or may be exogenous gamma immunoglobulins which have been administered to a subject prior to the method of the invention being carried out - including any therapeutic IgG molecule of any origin. Inactivation of serum IgG typically means a reduction in the Fc receptor interaction of IgG molecules. The term “Fc receptor” refers to Fc gamma immunoglobulin receptors (FcyRs) which are present on cells. In humans, FcyR refers to one, some, or all of the family of receptors comprising FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIC (CD32C), FcyRIIIA (CD 16a) and FcyRIIIB (CD 16b). As used herein, the term FcyR includes naturally occurring polymorphisms of FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIC (CD32C), FcyRIIIA (CD16a) and FcyRIIIB (CD16b).

The enzyme used in the method of the invention may be any enzyme which inactivates serum IgG, but is typically an IgG cysteine protease which cleaves IgG such that the antigen binding domains and Fc interacting domains are separated from each other. In such cases, Fc receptor interaction of serum IgG molecules is reduced because the quantity of intact IgG molecules in the serum is reduced. As another example, the enzyme may be an IgG endoglycosidase which cleaves a glycan structure on the Fc interacting domain of IgG, particularly the N-linked bi-antennary glycan at position Asn-297 (Kabat numbering). This glycan structure has a critical role in Fc receptor binding and complement activation. Thus, when it is wholly or partially removed by a protein, this will lead to reduced Fc receptor binding or complement activation by an otherwise intact IgG molecule, as well as also reduced recycling/half-life due to reduced binding to the FcRn. Enzymes suitable for use in the conditioning regimen are discussed in more detail in subsequent sections below. The enzyme is preferably administered by intravenous infusion, but may be administered by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes. The amount of the enzyme that is administered may be between 0.01 mg/kg BW and 2 mg/kg BW, between 0.01 mg/kg BW and 1 mg/kg BW, between 0.01 mg/kg BW and 0.5 mg/kg BW, between 0.01 and 0.3 mg/kg BW, preferably between 0.1 and 0.3 mg/kg BW, most preferably between 0.2 and 0.3 mg/kg BW. Doses of approximately 0.12 mg/kg BW, approximately 0.24 mg/kg BW and 0.5 mg/kg BW have been used in clinical settings. A particularly preferred dose is approximately 0.25 mg/kg BW.

The enzyme may be administered on multiple occasions to the same subject, provided that the quantity of anti-drug antibody (ADA) in the serum of the subject which is capable of binding to the enzyme does not exceed a threshold determined by the clinician. The amount of enzyme administered may be increased should a clinician consider this to be appropriate. The quantity of ADA in the serum of the subject which is capable of binding to the protease may be determined by any suitable method, such as an agent specific CAP FEIA (ImmunoCAP) test or a titre assay. If ADA in the subject exceed said threshold, the condition regimen may include administration of an alternative enzyme.

The enzyme may be any of the following:

IgG cysteine proteases

The IgG cysteine protease for use with the invention is specific for IgG. In preferred embodiments, the protease for use in the methods of the invention is IdeS (Immunoglobulin G-degrading enzyme of S. pyogenes), otherwise known as imlifidase. IdeS is an extracellular cysteine protease produced by the human pathogen S. pyogenes. IdeS was originally isolated from a group A Streptococcus strain of serotype Ml, but the ides gene has now been identified in all tested group A Streptococcus strains. IdeS has an extraordinarily high degree of substrate specificity, with its only identified substrate being IgG. IdeS catalyses a single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. IdeS also catalyses an equivalent cleavage of the heavy chains of some subclasses of IgG in various animals. IdeS efficiently cleaves IgG to Fc and F(ab’)2 fragments via a two-stage mechanism. In the first stage, one (first) heavy chain of IgG is cleaved to generate a single cleaved IgG (scIgG) molecule with a non-covalently bound Fc/2 molecule. The scIgG molecule is effectively an intermediate product which retains the remaining (second) heavy chain of the original IgG molecule. In the second stage of the mechanism this second heavy chain is cleaved by IdeS to release a F(ab’)2 fragment and a homodimeric Fc fragment. These are the products generally observed under physiological conditions. Under reducing conditions the F(ab’)2 fragment may dissociate to two Fab’ fragments and the homodimeric Fc may dissociate into its component monomers. IdeS has been shown to be particularly effective at cleaving IgG in humans. The entire plasma IgG- pool is cleaved within minutes of dosing with IdeS, and IgG levels in blood remain low for more than a week until newly synthesized IgG appeared in plasma. This demonstrates that the entire extracellular IgG pool and not only the plasma pool (i.e. serum IgG molecules) is cleaved by IdeS (Winstedt et al; PloS One 2015; 10(7): e0132011).

SEQ ID NO: 1 is the full sequence of IdeS including the N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP 010922160.1. SEQ ID NO: 2 is the mature sequence of IdeS, lacking the N terminal methionine and signal sequence. It is also available as Genbank accession no. ADF13949.1.

In alternative embodiments, the protease for use in the methods of the invention is IdeZ, which is a IgG cysteine protease produced by Streptococcus equi ssp. Zooepidemicus. a bacterium predominantly found in horses. SEQ ID NO: 3 is the full sequence of IdeZ including N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP 014622780.1. SEQ ID NO: 4 is the mature sequence of IdeZ, lacking the N terminal methionine and signal sequence.

In alternative embodiments, the protease for use in the methods of the invention is a hybrid IdeS/Z, such as that of SEQ ID NO: 5. The N terminus is based on IdeZ lacking the N terminal methionine and signal sequence.

In preferred embodiments, the protease for use in the invention may comprise or consist of SEQ ID NO: 2, 4 or 5. Proteases for use in the invention may comprise an additional methionine (M) residue at the N terminus and/or a tag at the C terminus to assist with expression in and isolation from standard bacterial expression systems. Suitable tags include a histidine tag which may be joined directly to the C terminus of a polypeptide or joined indirectly by any suitable linker sequence, such as 3, 4 or 5 glycine residues. The histidine tag typically consists of six histidine residues, although it can be longer than this, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2 or 1 amino acids.

In further preferred embodiments, the protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 6 to 25. These sequences represent IdeS and IdeZ polypeptides with increased protease activity and/or reduced immunogenicity. Each of SEQ ID NOs: 6 to 25 may optionally include an additional methionine at the N terminus and/or a histidine tag at the C terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C terminus by a linker of 3x glycine or 5x glycine residues.

In further preferred embodiments, the protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 56 to 69. These sequences represent IdeS polypeptides with increased protease activity and/or reduced immunogenicity. Each of SEQ ID NOs: 56 to 69 may optionally include an additional methionine at the N terminus and/or a histidine tag at the C terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C terminus by a linker of 3x glycine or 5x glycine residues.

In further preferred embodiments, the protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 6 to 25, optionally with up to 3 (such as 1, 2 or 3) amino acid substitutions. Each of SEQ ID NOs: 6 to 25 and variants thereof may optionally include an additional methionine at the N terminus and/or a histidine tag at the C terminus.

In further preferred embodiments, the protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 56 to 69, optionally with up to 3 (such as 1, 2 or 3) amino acid substitutions. Each of SEQ ID NOs: 56 to 69 and variants thereof may optionally include an additional methionine at the N terminus and/or a histidine tag at the C terminus.

The polypeptide of the invention is typically at least 100, 150, 200, 250, 260, 270, 280, 290, 300 or 310 amino acids in length. The polypeptide of the invention is typically no larger than 400, 350, 340, 330, 320 or 315 amino acids in length. It will be appreciated that any of the above listed lower limits may be combined with any of the above listed upper limits to provide a range for the length the polypeptide of the invention. For example, the polypeptide may be 100 to 400 amino acids in length, or 250 to 350 amino acids in length. The polypeptide is preferably 290 to 320 amino acids in length, most preferably 300 to 315 amino acids in length.

The primary structure (amino acid sequence) of a protease of the invention is based on the primary structure of IdeS, IdeZ or IdeS/Z, specifically the amino acid sequence of SEQ ID NO: 2, 4 or 5, respectively. The sequence of a protease of the invention may comprise a variant of the amino acid sequence of SEQ ID NO: 2, 4 or 5, which is at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 4 or 5. The variant sequence may be at least 80%, at least, 85%, preferably at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence of SEQ ID NO: 2, 4 or 5. The variant may be identical to the sequence of SEQ ID NO: 2, 4 or 5 apart from the inclusion of one or more of the specific modifications identified in WO2016/128558 or WO2016/128559. Identity relative to the sequence of SEQ ID NO: 2, 4 or 5 can be measured over a region of at least 50, at least 100, at least 200, at least 300 or more contiguous amino acids of the sequence shown in SEQ ID NO: 2, 4 or 5, or more preferably over the full length of SEQ ID NO: 4 or 5.

The protease for use in the invention may be an IdeS, IdeZ or IdeS/Z polypeptide that comprises a variant of the amino acid sequence of SEQ ID NO: 2, 4 or 5 in which modifications, such as amino acid additions, deletions or substitutions are made relative to the sequence of SEQ ID NO: 2, 4 or 5. Such modifications are preferably conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art.

IgG cysteine protease activity may be assessed by any suitable method, for example by incubating a polypeptide with a sample containing IgG and determining the presence of IgG cleavage products. Suitable methods are described in the WO2016/128559. Suitable assays include an ELISA-based assay, such as that which is described in WO2016/128559. In such an assay, the wells of an assay plate will typically be coated with an antibody target, such as bovine serum albumin (BSA). Samples of the polypeptide to be tested are then added to the wells, followed by samples of target-specific antibody that is antibody specific for BSA in this example. The polypeptide and antibody are allowed to interact under conditions suitable for IgG cysteine protease activity. After a suitable interval, the assay plate will be washed and a detector antibody which specifically binds to the target-specific antibody will be added under conditions suitable for binding to the target-specific antibody. The detector antibody will bind to any intact target-specific antibody that has bound to the target in each well. After washing, the amount of detector antibody present in a well will be proportional to the amount of target-specific antibody bound to that well. The detector antibody may be conjugated directly or indirectly to a label or another reporter system (such as an enzyme), such that the amount of detector antibody remaining in each well can be determined. The higher the potency of the tested polypeptide that was in a well, the less intact target-specific antibody will remain and thus there will be less detector antibody. Typically, at least one well on a given assay plate will include IdeS instead of a polypeptide to be tested, so that the potency of the tested polypeptides may be directly compared to the potency of IdeS. IdeZ and IdeS/Z may also be included for comparison.

Other assays may determine the potency of a tested polypeptide by directly visualizing and/or quantifying the fragments of IgG which result from cleavage of IgG by a tested polypeptide. An assay of this type is also described in WO2016/128559. Such an assay will typically incubate a sample of IgG with a test polypeptide (or with one or more of IdeS, IdeZ and IdeS/Z as a control) at differing concentrations in a titration series. The products which result from incubation at each concentration are then separated using gel electrophoresis, for example by SDS-PAGE. Whole IgG and the fragments which result from cleavage of IgG can then be identified by size and quantified by the intensity of staining with a suitable dye. The greater the quantity of cleavage fragments, the greater the potency of a tested polypeptide at a given concentration. A polypeptide of the invention will typically produce detectable quantities of cleavage fragments at a lower concentration (a lower point in the titration series) than IdeZ and/or IdeS. This type of assay may also enable the identification of test polypeptides that are more effective at cleaving the first or the second heavy chain of an IgG molecule, as the quantities of the different fragments resulting from each cleavage event may also be determined. A polypeptide of the invention may be more effective at cleaving the first chain of an IgG molecule than the second, particularly when the IgG is an IgG2 isotype. A polypeptide of the invention may be more effective at cleaving IgGl than IgG2.

IgG endoglycosidases

The enzyme may have IgG endoglycosidase activity, preferably cleaving the glycan moiety at Asn-297 (Kabat numbering) in the Fc region of IgG. An example of such a protein is EndoS (Endoglycosidase of S. pyogenes). EndoS hydrolyzes the -1,4-di-A- acetylchitobiose core of the asparagine-linked glycan of normally-glycosylated IgG. The mature sequence of EndoS is provided as SEQ ID NO: 90. The agent may be a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 90, or may be a homologue thereof from an alternative bacterium, such as Streptococcus equi or Streptococcus zooepidemicus, or Coryn ebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. The agent may be CP40, EndoE, or EndoF2. Alternatively the agent may be a variant of the EndoS protein which comprises or consists of any amino acid sequence which has at least 80%, 85%, 90% or 95% identity with SEQ ID NO: 90 and has IgG endoglycosidase activity. A variant of the EndoS protein may comprise or consist of an amino acid sequence in which up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, amino acid substitutions, insertions or deletions have been made relative to the amino acid sequence of SEQ ID NO: 90, provided the variant has IgG endoglycosidase activity. Said amino acid substitutions are preferably conservative. Conservative substitutions are as defined in the preceding section.

Alternatively the agent may be a protein which comprises or consists of a fragment of SEQ ID NO: 90 and has IgG endoglycosidase activity, preferably wherein said fragment is 400 to 950, 500 to 950, 600 to 950, 700 to 950 or 800 to 950 amino acids in length. A preferred fragment consists of amino acids 1 to 409 of SEQ ID NO: 90, which corresponds to the enzymatically active a-domain of EndoS generated by cleavage by the streptococcal cysteine proteinase SpeB. The fragment may be created by the deletion of one or more amino acid residues of the amino acid sequence of SEQ ID NO: 90. Up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 550 residues may be deleted, or more. The deleted residues may be contiguous with other.

Any fragment or variant of SEQ ID NO: 90 preferably includes residues 191 to 199 of SEQ ID NO: 90, i.e. Leu-191, Asp- 192, Gly-193, Leu- 194, Asp- 195, Vai- 196, Asp- 197, Val- 198 and Glu-199 of SEQ ID NO: 90. These amino acids constitute a perfect chitinase family 18 active site, ending with glutamic acid. The glutamic acid in the active site of chitinases is essential for enzymatic activity. Most preferably, therefore, a variant of SEQ ID NO: 90 contains Glu-199 of SEQ ID NO: 90. The variant of SEQ ID NO: 90 may contain residues 191 to 199 of SEQ ID NO: 90 having one or more conservative substitutions, provided that the variant contains Glu-199 of SEQ ID NO: 90.

Conditioning regime — dose and timing for lymphodepletion and enzymatic inactivation

The dose and timing of administration of a lymphodepleting agent comprising IgG molecules in accordance with step (a) of the method will typically be such that lymphocyte number and/or activity is substantially down-modulated in the individual subject. Suitable dose schedules for known lymphodepleting agents are well known in the art. The agent may be administered as one or more doses, administered spaced out over one or more days. The first dose may be administered at any time, provided the final dose is administered at least one day prior to step (c). The day prior to step (c) may be referred to as D -1, with the day of transplant referred to as D 0. A typical schedule may have a first dose of agent taking place around 14, 13, 12, 11, 10, 8, 7, 6, 5, 4 or 3 days prior to step (c) (may be referred to as D -14, D -13, D -12 etc) followed by, for example, 2 to 7 (for a total of 3 to 8) or 2 to 5 (for a total of 3 to 6) separate, additional doses, typically one dose per day, provided that the final dose is administered at least one day prior to step (c), that is no later than D -1.

The inventors have surprisingly discovered that the methods of the invention work particularly well where a subject has initially received a higher dose of the lymphodepleting agent. Thus, in a preferred embodiment, step (a) uses a higher dose of the lymphodepleting agent. Suitable doses will vary between different agents and will be known to a skilled person. For example, where the agent is an anti-CD52 antibody (such as alemtuzumab) the total dose may be l-20mg/kg, 5-20 mg/kg, or 10-20 mg/kg. Where the agent is a rATG such as ATG-G (Grafalon) the total dose may be 30-150 mg/kg, 60-150 mg/kg, 100-150 mg/kg. Where the agent is a rATG such as Thymoglobulin, the total dose may be 10-50 mg/kg, 20-50 mg/kg, 30-50 mg/kg. In other embodiments, a low dose of the agent may be administered. For example, where the agent is an anti-CD52 antibody (such as alemtuzumab) the dose may be 0.005-lmg/kg, 0.005-0.1 mg/kg, or 0.005-0. Olmg/kg. Where the agent is a rATG such as ATG-G (Grafalon) the dose may be 5-30 mg/kg, 5-20 mg/kg, or 5-10 mg/kg. Where the agent is an anti-thymoglobulin antibody the dose may be 0.5-10 mg/kg, 0.5 - 3 mg/kg, or 0.5 - 1 mg/kg.

The IgG inactivating enzyme is administered in step (b) after the lymphodepleting agent in step (a), typically at a time point sufficiently after step (a) that the lymphodepleting agent has had the desired effects on lymphocyte numbers and/or activity, but sufficiently in advance of the subsequent cell transplant that inactivation by the enzyme will prevent negative effects that may be caused by the agent. The timing of step (b) is typically at least 1 day after the conclusion of step (a), that is 1 day after the final dose of agent is administered in step (a). Step (b) may take place on the same day as step (c), that is on D 0, but may preferably be at least 2, 3, 4, 5 or 6 hours prior to step (c), also preferably, step (b) may take place at least 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4, 5, 6, 7, 8, 9 or 10 days prior to step (c). The dose and timing of administration of the IgG inactivating enzyme will typically be sufficient to inactivate substantially all serum IgG molecules in the individual subject (i.e. including all of the lymphodepleting agent comprising IgG molecules), prior to administration of the transplant in step (c). Suitable dose schedules for known IgG inactivating enzymes are well known in the art. The timing of step (c) is particularly relevant because the F(ab’)2 fragments which result from the cleavage of the IgG molecule can retain some of the cytotoxic activity of the uncleaved antibody. Thus, generally, it is desirable to leave sufficient time (e.g. 0-6 days, for example 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4 days, 5 days, 6 days or more than 72 hours) for these F(ab’)2 to clear before the transplant is administered. Surprisingly, the inventors have shown that the F(ab’)2 fragments stemming from cleavage of an anti- CD52 antibody (e.g. alemtuzumab) according to the invention does not show this cytotoxic effect. The methods of the invention thus work particularly well with such antibodies. In embodiments that use such an anti-CD52 antibody, step (b) may be carried out less than 24 hours (e.g. less than 12 hours, less than 6 hours, less than 2 hours, less than 1 hour, less than 30 minutes or less than 15 minutes) before step (c).

The methods of the invention do allow the depletion of cells well before the transplant. However, if the enzyme that is administered to deplete the therapeutic antibodies was administered too long before transplantation there is a risk that the depleted lymphocytes will recover. A skilled person would therefore understand that the gap between steps (b) and (c) should not exceed the time for the recipients cells to reappear.

By way of illustration, it is preferred that: where the lymphodepleting agent is alemtuzumab, the inactivating enzyme should be administered at a time and dose such that the level of intact agent present in the subject immediately prior to transplant is reduced to less than 0. Ipg/ml; where the lymphodepleting agent is ATG-G, the inactivating enzyme should preferably be administered at a time and dose such that the level of intact agent present in the subject immediately prior to transplant is reduced to less than Ipg/ml; and where the lymphodepleting agent is Thymoglobulin, the inactivating enzyme should preferably be administered at a time and dose such that the level of intact agent present in the subject immediately prior to transplant is reduced to less than 0.3pg/ml.

Dose and timing schedules for each of various exemplary lymphodepleting agents comprising IgG molecules, and an exemplary IgG inactivating enzyme, are shown below to illustrate the invention. Timing is shown relative to the day of transplant in step (c). As discussed above, in preferred embodiments, the lymphodepleting agent is administered at a high dose.

A particular exemplary dose and timing schedule for each of the various exemplary lymphodepleting agents comprising IgG molecules and an exemplary IgG inactivating enzyme is shown below. Timing is shown relative to the day of cell transplant.

Conditioning regimen — additional steps

The conditioning regimen may additionally comprise one or more of:

(i) administration to the subject of a non-lethal dose of irradiation and/or any agent which depletes the subject’s HSPC;

(ii) administration any other agent or regimen which modulates (e.g. reduces) the activity of the immune system, e.g., inhibitors of complement, inhibitors of cytokines, inhibitors of innate immune cells, inducers of tolerance.

Step (i) typically involves administering a dose of radiation which is sufficient to partially or totally eradicate (or ablate) the bone marrow of the subject. It is particularly applicable when the donor cells are HSPCs. Partial eradication by irradiation may be preferred since the side effects are typically less severe and also because it is desirable to retain some recipient bone marrow. The ablation of recipient bone marrow creates space in the bone marrow for engraftment of donor cells, but also depletes lymphocytes in the subject and thus also reduces immune system activity in the same manner as step (a). As such, the conditioning regimen preferably includes at least (a), but most preferably includes at least (a) and (i). Alternatively, it may be preferred in step (a) to use an irradiation free approach to depletion of subject HSPCs, such as administration of anti-CDl 17 and/or anti-CD47. This will create space for engraftment of donor HSPCs, but without some of the undesirable side- effects of irradiation. In addition to HSPC depletion, the subject may also optionally receive an infusion of donor CD8-alpha cells, which may increase the frequency of stable chimerism in sensitized recipients. Donor T cell infusion may promote donor HSPC engraftment by reducing survival of host T cells.

Method for inducing hematopoietic chimerism

The present invention provides a method for the induction of hematopoietic chimerism in a subject, the method comprising conducting the conditioning regimen of the invention and subsequently administering HSPC to the subject in an amount sufficient and under conditions suitable to induce hematopoietic chimerism in the subject. The method may alternatively be described as a method for the stable transplantation of HSPC. The HSPC may be autologous (the patient's own cells are used) or syngeneic (the cells are from a genetically identical twin), or they may allogeneic (the cells come from a separate, nonidentical donor).

Immune complications which reduce the likelihood of successful engraftment of HSPC in the recipient are most significant for allogeneic cells and thus the method of the invention is of greatest benefit with such cells. However, immune complications can occur even with autologous cells if there is expression of a product to which the recipient has not previously been exposed. If an autologous cell has been genetically modified to express a gene therapy, the cell may be sufficiently altered to provoke an immune response. For example there may be an immune response to the expressed gene therapy product. Similar would apply if the HSPC has been genetically modified to express a different HLA type which is not matched to the HLA of the recipient. Therefore the HSPC are preferably allogeneic, or are genetically modified autologous or syngeneic cells, for example Chimeric antigen receptor (CAR) T-cells. The HSPC are most preferably allogeneic. In a particularly preferred embodiment, the HSPC are from a donor who is also the donor of another organ or tissue which is to be transplanted into the recipient. That is, the same donor provides both the HSPC and the other cell, organ or tissue.

HSPC are found in the bone marrow of adults, especially in the pelvis, femur, and sternum. They are also found in umbilical cord blood and, in small numbers, in peripheral blood. HSPC may be harvested from these locations using any suitable technique established in the art.

For example, HSPC may be harvested from human bone marrow by aspirating directly from the centre of a bone of the donor with a large needle. The posterior iliac crest is the usual site of harvest. The technique is referred to as a bone marrow harvest and may be performed under local or general anesthesia. When the administered HSPC are derived from the bone marrow of the donor, the administration of HSPC may be described as a bone marrow transplant (BMT).

HSPC may be harvested from umbilical cord blood shortly after the birth of an infant. The umbilical cord is double-clamped from the umbilicus and transacted between clamps. The umbilical cord vein is then punctured under sterile conditions, and the blood flows freely by gravity into an anti coagulated sterile closed harvesting system, form which the HSPC may be isolated.

HSPC may be harvested from peripheral blood, typically by apheresis. However, because numbers of HSPC in peripheral blood are normally low, it is first necessary to mobilize HSPCs from the bone marrow. In a healthy donor, this can be achieved by administration of Granulocyte colony-stimulating factor (G-CSF). Alternative strategies may be required if the donor is not healthy. This may frequently be the case if the intended HSPC transplant is autologous.

HSPC are preferably used as quickly as possible after harvesting (that is fresh), but may be cryopreserved for storage prior to thawing for use in the method of the invention. Cryopreservation typically includes volume depletion by removal of red cells and plasma. The quantity of stem cells in the harvest may be quantified, e.g. by flow cytometric analysis of a sample, to establish the proportion of cells which are positive for CD34 (a marker for stem cells).

The HSPC may be administered to the subject by any suitable method. A preferred method is infusion, typically through a central line. The patient may be kept in highly clean or sterile conditions, such as in a room with high-efficiency particulate air (HEP A) filters under positive pressure, before, during and after the infusion to reduce the risk of infection.

The method may be monitored to determine that the HSPC transplant has successfully resulted in hematopoietic chimerism. This is achieved by determining the proportion of donor-derived hematopoietic cells present in a blood sample taken from the subject after a particular time interval, typically 28 days after administration of the HSPC. For example, hematopoietic chimerism may be defined as achieved if at least 5% of the lymphocytes and/or myeloid cells in the sample are found to be donor-derived, preferably if at least 5% of the lymphocytes in the sample are found to be donor-derived. The chimerism is described as mixed if no more than 90% of the lymphocytes and/or myeloid cells in the sample are found to be donor-derived (that is at least 10% are still derived from the recipient), preferably if no more than 90% of the lymphocytes in the sample are found to be donor-derived (that is at least 10% of lymphocytes are still derived from the recipient). The chimerism may be described as total if 98% or more of the lymphocytes and/or myeloid cells in the sample are found to be donor-derived. Mixed chimerism is typically preferred for the methods of the invention, because the recipient will have a greater level of immunocompetence. However, full chimerism may be beneficial in some circumstances, for example in the treatment of cancers such as leukemia where the goal is to eliminate host cells with the potential to cause cancer recurrence, replacing them with the transplanted HSPC.

The proportion of donor and recipient derived cells in a sample may be determined by any suitable method in the art, such as flow cytometric analysis as described in the Examples. Real-time PCR may also be used. Other methods are discussed in Agrawal et al Bone Marrow Transplantation 2004 (34) p-12.

Methods of treating or preventing a disease or condition

The present invention provides a method for the prevention or treatment of a disease or condition in a subject. The method comprises administering a cell transplant to the subject, wherein said administering comprises the conditioning regimen of the invention. Expressed another way, the invention also provides a method for the prevention or treatment of immune rejection of a cell transplant, the method comprising the conditioning regimen of the invention.

The cell transplanted may be of any type, including HSPC. The HSPC may be a genetically modified HSPC, such that the method effectively comprises administering a gene therapy to the subject in which the HSPC is the vector. The cell to be transplanted may originate from a different species to the recipient, that is it may be a xenotransplant. Suitable species for xenotransplantation into human recipients may include pigs or non-human primates. In such cases the HSPC may be genetically modified to aid with tolerance to the transplant. The cell that is a xenotransplant may also be genetically modified.

The subject to be treated may be sensitized or highly sensitized. By “sensitized” it is meant that the subject has developed antibodies to human major histocompatibility (MHC) antigens (also referred to as human leukocyte antigens (HLA)). The anti-HLA antibodies originate from allogeneically sensitized B-cells and are usually present in patients that have previously been sensitized by blood transfusion, previous transplantation or pregnancy. Achieving hematopoietic chimerism in sensitized patients may reverse allosensitization, through the generation of specific tolerance in T and B cells, resulting in a reduction of donor specific immune responses such as DSA.

Whether or not a potential transplant recipient is sensitized may be determined by any suitable method. For example, a Panel Reactive Antibody (PRA) test may be used to determine if a recipient is sensitized. A PRA score >30% is typically taken to mean that the patient is “high immunologic risk” or “sensitized”. Alternatively, a cross match test may be conducted, in which a sample of the potential transplant donor’s blood is mixed with that of the intended recipient. A positive cross-match means that the recipient has antibodies which react to the donor sample, indicating that the recipient is sensitized and transplantation should not occur. Cross-match tests are typically conducted as a final check immediately prior to transplantation.

The method may be for the prevention or treatment of any disease or condition that is treated by HSPC transplant. Diseases or conditions typically treated by HSPC transplant may be acquired or congenital. Acquired diseases or conditions that may be treated by HSPC transplant include:

- Hematological malignancies such as leukemias (for example Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML); lymphomas (for example Hodgkin's disease, NonHodgkin's lymphoma) and Myelomas (for example, Multiple myeloma (Kahler's disease)).

- Solid tumor cancers, such as Neuroblastoma, Desmoplastic small round cell tumor, Ewing's sarcoma, Choriocarcinoma.

- Hematologic diseases such as phagocyte disorders (for example Myelodysplasia); Anemias (for example Paroxysmal nocturnal hemoglobinuria (PNH; severe aplasia), Aplastic anemia, Acquired pure red cell aplasia); Myeloproliferative disorders (for example Polycythemia vera, Essential thrombocytosis, Myelofibrosis).

- Metabolic disorders such as amyloidosis (for example Amyloid light chain (AL) amyloidosis).

- Environmentally-induced diseases such as radiation poisoning.

- Viral diseases such as Human T-lymphotropic virus (HTLV) or Human Immunodeficiency Viruses (HIV).

- Autoimmune diseases, such as multiple sclerosis.

Congenital diseases or conditions that may be treated HSPC transplant include:

- Lysosomal storage disorders such as Lipidoses - disorders of lipid storage - (for example Neuronal ceroid lipofuscinoses, Infantile neuronal ceroid lipofuscinosis (INCL, Santavuori disease,), Jansky-Bielschowsky disease (late infantile neuronal ceroid lipofuscinosis)); Sphingolipidoses (for example Niemann-Pick disease, Gaucher disease); Leukodystrophies (for example Adrenoleukodystrophy, Metachromatic leukodystrophy, Krabbe disease (globoid cell leukodystrophy)); Mucopolysaccharidoses (for example Hurler syndrome (MPS I H, a-L-iduronidase deficiency), Scheie syndrome (MPS I S), Hurler- Scheie syndrome (MPS I H-S), Hunter syndrome (MPS II, iduronidase sulfate deficiency), Sanfilippo syndrome (MPS III), Morquio syndrome (MPS IV), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII)); Glycoproteinoses (for example Mucolipidosis II (I-cell disease), Fucosidosis, Aspartylglucosaminuria, Alpha-mannosidosis; or Others (for example Wolman disease (acid lipase deficiency)

- Immunodeficiencies, such as T-cell deficiencies (for example Ataxia-telangiectasia, DiGeorge syndrome); Combined T- and B-cell deficiencies (for example Severe combined immunodeficiency (SCID), all types); Wiskott-Aldrich syndrome; Phagocyte disorders (for example Kostmann syndrome, Shwachman-Diamond syndrome); Immune dysregulation diseases (for example Griscelli syndrome, type II); Innate immune deficiencies (for example NF-Kappa-B Essential Modulator (NEMO) deficiency

- Hematologic diseases, such as Hemoglobinopathies (for example Sickle cell disease, P thalassemia major (Cooley's anemia)); Anemias (for example Aplastic anemia, Diamond-Blackfan anemia, Fanconi anemia); Cytopenias (for example Amegakaryocytic thrombocytopenia); and Hemophagocytic syndromes (for example Hemophagocytic lymphohistiocytosis (HLH)).

Where the HSPC are genetically modified to administer a gene therapy, the method of the invention may be for the prevention or treatment of the di sease or condition to which said gene therapy is directed.

The invention also provides an enzyme which inactivates serum IgG molecules in a subject for use in a method for the prevention or treatment of a disease or condition, wherein the method is as described above.

The invention also provides the use of an enzyme which inactivates serum IgG molecules in a subject in the manufacture of a medicament, wherein the medicament is for the prevention or treatment of a disease or condition in a method as described above.

In some embodiments, the cell transplanted may be a chimeric antigen receptor (CAR) cell, such as a CAR-T cells, CAR-NK cell, CAR-B-cells, CAR macrophage etc. These embodiments are particularly useful in the treatment of cancers, such as a hematological cancers (for example leukemia), lymphoma, myeloma, virus infections (such as HIV) and autoimmune diseases.

In other embodiments, the cell transplanted is a transfused or endogenous platelet cell.

Production of polypeptides

The enzymes used in the methods of the invention are polypeptides and may be produced by any suitable means. For example, a polypeptide may be synthesised directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry or by solution phase peptide synthesis. Alternatively, a polypeptide may be produced by transforming a cell, typically a bacterial cell, with a nucleic acid molecule or vector which encodes said polypeptide. Production of enzyme polypeptides by expression in bacterial host cells is described and exemplified in WO2016/128558 and WO2016/128559.

Compositions and formulations comprising polypeptides

The present invention also provides compositions comprising an enzyme for use in the methods of the invention. For example, the invention provides a composition comprising one or more polypeptides, and at least one pharmaceutically acceptable carrier or diluent. The carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the composition and not deleterious to a subject to which the composition is administered. Typically, carriers and the final composition are sterile and pyrogen free.

Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan. For example, the enzyme can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, reducing agents and the like, may be present in the excipient or vehicle. Suitable reducing agents include cysteine, thioglycerol, thioredoxin, glutathione and the like. Excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi -dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally- acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.

Other parenterally-administrable compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. The compositions may be suitable for administration by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes. Preferred compositions are suitable for administration by intravenous infusion.

Kits

The invention also provides a kit for carrying out the methods described herein. The kit of the invention may include an enzyme or a composition comprising an enzyme, as described above. The kit may include means for administering the enzyme or composition to a subject. The kit may include instructions for use of the various components in any method as described herein.

EXAMPLES

Unless indicated otherwise, the methods used are standard biochemistry and molecular biology techniques. Examples of suitable methodology textbooks include Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley and Sons, Inc.

Example 1 - optimal spacing of imlifidase and rATG

Background

Imlifidase (conditionally authorised in the EU for kidney transplant desensitization) is a cysteine protease which cleaves all subclasses of human and rabbit IgG to a F(ab’)2 fragment and a dimeric Fc fragment. Rabbit anti -thymocyte globulin (rATG) is a depleting antibody therapy approved for induction in kidney transplantation (it effects a large reduction in circulating T-lymphocytes). Antibody-based therapies such as rATG may be inactivated if given with imlifidase. The purpose of this study was to investigate the cleavage of rATG by imlifidase.

Methods

The cleavage pattern of rATG was investigated with sera from healthy subjects (n=l 1) treated with 0.25 mg/kg imlifidase (EudraCT number: 2019-002770-31). Serum samples were incubated with a fixed, clinically relevant, concentration of 50 pg/mL rATG (commonly observed after a dose of 1.5 mg/kg), for 2 hours at 37°C. Serum samples were collected pre- imlifidase through 14 days post-imlifidase and were analyzed using SDS-PAGE and Western blot, developed with a goat anti-rabbit IgG, F(ab’)2 specific antibody to evaluate the cleavage of rATG. Imlifidase concentration was analyzed using a validated electroluminescence immunoassay based on MSD technology.

Results

The imlifidase serum concentration in the subjects declined rapidly and at 96 hours the mean concentration was 0.5 pg/mL, though with a large individual variation, <0.1-1.8 pg/mL (Figure 1). At this timepoint the level of imlifidase activity had decreased sufficiently to avoid complete cleavage of rATG in 8 of 11 subjects (Figure 2).

Conclusions rATG is rapidly cleaved and inactivated by imlifidase in human serum.

Example 2 - use of imlifidase in preconditioning regimens with different antibodybased lymphodepleting agents

Cell therapies such as hematopoietic stem cell transplantation (HSCT) can be curative treatment options for patients with malignant diseases, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and for patients with non-malignant diseases, e.g., immune deficiencies, Thalassemia and sickle cell disease. rATG agents (such as Grafalon (ATG-G) or Thymoglobulin) and anti-CD52 antibodies such as alemtuzumab are used prior to allogeneic T-cell depleted stem cell transplantation to allow a reduced intensity conditioning, leading to a significant reduction of graft versus host disease (GvHD) and engraftment failure. High doses of these drugs, however, are associated with delayed immune recovery due to their long half-life and will enhance the risk of relapse and infection. Therefore, a means to protect donor lymphocytes, particularly donor NK cells, from any remaining lymphodepleting antibodies may be beneficial.

Imlifidase is a cysteine protease derived from Streptococcus pyogenes which effectively hydrolyzes human and rabbit IgG, in the lower hinge region, into a F(ab’)2 fragment and a dimeric Fc/2-fragment (2*Fc/2). The resulting F(ab’)2 fragment retains full binding capacity to its antigens but the IgG-dependent Fc-dependent effector mechanisms, such as cell-mediated toxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are inactivated.

Since the mechanisms of action of ATG-G, Thymoglobulin and alemtuzumab are, to a large extent, Fc-mediated, imlifidase (or similar enzymes) may be used as a tool to inactivate these drugs and eliminate toxicity, especially towards NK cells, before HSCT. Such a reduced intensity conditioning practice can be particularly useful in more vulnerable patients such as young children and elderly. In this case, imlifidase-inactivated high doses of ATG-G, Thymoglobulin and alemtuzumab, leads to reduction of risk of GvHD, engraftment failure and relapse while boosting immune recovery by eliminating toxicity towards NK cells thus fostering a quicker immune recovery. The suitability of imlifidase (or similar enzymes) in this context is demonstrated by the experiments performed here. Materials and Methods

SDS-PAGE analysis of antibody cleavage by imlifidase

ATG-G, alemtuzumab, ATGAM, and thymoglobulin (1 mg/mL) were diluted in culture medium and incubated with 20 pg/mL imlifidase at 37°C for 2 hours. The samples were diluted lOx in PBS and further diluted in 4X SDS sample buffer and incubated at 92°C for 3 min. The samples were run on 4-20% Mini-PROTEAN® tgx PRECAST SDS-PAGE gels (200V, 40 min), using lx Tris/Glycine/SDS-buffer. As size standard, 5 pL Precision Plus MW standard (#161-0363 Bio-Rad) was used. The gels were activated for 2.5 min and exposed for 1 s using a Gel Doc EZ Imager system (Bio-Rad) and Image Lab 6.0 software (Bio-Rad).

Preparation of F(ab’)2 and 2*Fc/2-fragments for rATG and alemtuzumab rATG and alemtuzumab were hydrolyzed, using the FragIT Kit containing immobilized IdeS coupled to agarose beads on spin-columns, for 45 min at 37°C on a shaker. The antibody fragments were collected after centrifugation and transferred to rabbit- or human-specific Fc-binding spin-columns to separate Fc- and F(ab‘)2-fragments. The concentrations of the fragment fractions were determined by NanoDrop.

Cell lines used in the cytotoxicity assays

K562 is a target cell line from a CML patient in blast crisis carrying the BCR-ABL1 el4-a2 (b3-a2) fusion gene; The feeder cell line K562-mbIL15-4-lBBL express membrane- anchored IL15. Both cell lines are grown in RPMI 1640 medium with FCS (10%), L-Glut (2 mM) and Pen/Strep (100 pg/ml).

Cytotoxicity assay of full length rATG or alemtuzumab and their respective F(ab‘)i- 2*Fc/2- fragments on NK cells against K562 cells.

The expanded NK cells (eNKs) were generated by incubating PBMCs from healthy donors with IL-2 (50 U/ml) in the presence of feeder cells (K562-mbIL15-4-lBBL) for 14 days. These cells can be frozen in aliquots for posterior use in additional experiments. Thawed eNKs were cultivated for 16h in RPMI- 1640 medium supplemented with IL-2 (100 U/ml) and human AB serum (10%), then incubated with or without full length ATG-G or alemtuzumab or their respective F(ab’)2- or 2*Fc/2- fragments and human AB serum (25%) for 6h. These cells were subsequently used as effector (E) cells against calcein-labeled K562 wild type target cells (T) in calcein release assays for 2 hours on E:T, 10: 1 proportion. The amount of released calcein corresponds to the NK cell-mediated-cytotoxicity. Concentrations of F(ab’)2- and 2*Fc/2-fragments were chosen according to the percentage of molecular weight of full length alemtuzumab.

Specific lysis (%) was calculated as follows: [(test release minus spontaneous release)/(maximum release minus spontaneous release)] * 100. Spontaneous release represents the release of calcein from the target cells in medium alone and maximum release represents the calcein release from target cells lysed in medium plus 1% Triton X-100 (each measured in triplicates). Spontaneous release (%) was calculated as follows: [(spontaneous release minus background)/(maximum release minus background)] * 100. Background consists of medium plus the supernatant of calcein-labeled cells.

Alemtuzumab and Thymoglobulin CD3+ cytotoxicity in whole blood

Peripheral blood was collected from a healthy volunteer in sodium heparin tube (BD- Plymouth). The whole blood was incubated at 37°C, 5% CO2 for 4 and 16 hours with either alemtuzumab (Campath, anti-CD52, Genzyme) or Thymoglobulin (Genzyme), at final antibody concentrations of 100, 10, 1, 0.1 and 0 pg/mL. RBC were lysed upon sample incubation with Lysis Buffer (#349202, BD) for 5 minutes. The samples were centrifuged, and the supernatant discarded. The pellets were washed in FACS buffer and centrifuged at 1200 rpm for 3 min. The pellets were resuspended in 50 pL PE-conjugated anti-CD3 antibody (Cat. No. MAI-10179, Thermo Scientific) and incubated at 4°C for 30 mins. FACS- buffer was added and the samples were analyzed on Accuri C6 flow cytometer.

FACS and SDS-PAGE analysis of alemtuzumab cleavage by imlifidase in whole blood

Whole blood was incubated with 30, 3, 0.3, 0.03 and 0 pg/mL imlifidase for 6 hours at 37°C, 5% CO2. PBS/BSA or alemtuzumab (Campath, anti-CD52, Genzyme) were added to the samples at a final concentration of 100 pg/mL and incubated overnight at 37°C, 5% CO2. Part of these samples was directly used for SDS-PAGE where one pL plasma was mixed with 40 pL 2x SDS-loading buffer, heated for 5 min at 92°C, from which 5 pL were separated on a 4-20% SDS-PAGE gel (200 V, 40 min). Precision Plus MW standard, 5 pL, was used as control. The remaining of the samples were lysed and labelled with PE- conjugated anti-CD3 antibody (Cat. No. MAI-10179, Thermo Scientific) at 4°C for 30 mins and analyzed on Accuri C6 flow cytometer. FACS and SDS-PAGE analysis of alemtuzumab cleavage by imlifidase in whole blood.

Whole blood was incubated with imlifidase (30, 15, 7.5, 3.8, 1.9, 0.94, 0.47, 0.23, 0.12, 0.056 and 0 pg/mL) for 6 hours at 37°C, 5% CO2. PBS/BSA or alemtuzumab (Campath, anti-CD52, Genzyme) were added to the samples at a final concentration of 100 pg/mL and incubated overnight at 37°C, 5% CO2. Plasma aliquots of one pL were mixed with 40 pL 2x SDS-loading buffer, heated 5 min at 92°C, from which 5 pL were separated on a 4- 20% SDS-PAGE gel (200V, 40 min). Precision Plus MW standard, 5 pL, was used as control. The remaining of the samples were RBC-lysed and labelled with PE-conjugated anti- CD3 antibody (Cat. No. MAI-10179, Thermo Scientific) at 4°C for 30 mins and analyzed on Accuri C6 flow cytometer.

Efficacy test of horse ATGAM IgG after imlifidase treatment

Whole blood from a healthy donor was treated in vitro for 6 hours with imlifidase (30, 15,7.5, 3.8, 1.9, 0.94, 0.47, 0.23, 0.12, 0.059 and 0 pg/mL) before further incubation with 1% horse serum in PBS or 500 pg/mL ATGAM (equine Anti-thymocyte Globulin) for 16 hours at 37°C, 5% CO2. One pL sample aliquots were mixed with 40 pL 2x SDS-loading buffer, heated for 5 min at 92°C. Five pL were separated on a 4-20% SDS-PAGE gel (200V, 40 min). Precision Plus MW standard (5 pL) was used as control. The remaining of the ATGAM and imlifidase-treated samples were further RBC-lysed and labelled with PE-conjugated anti- CD3 antibody (Cat. No. MAI-10179, Thermo Scientific) at 4°C for 30 mins and analyzed on Accuri C6 flow cytometer.

Evaluating the blocking of CDC by alemtuzumab with eculizumab in a whole blood assay

Whole blood of a healthy donor was pre-treated with or without the anti-C5 eculizumab (Soliris) (200 pg/mL) and incubated at RT for 30 mins. The samples were further treated with either 30 pg/mL alemtuzumab (Campath, anti-CD52) or 200 pg/mL ATGAM for 4 hours at 37°C, 5% CO2, in the presence and absence of complement inhibitor. The samples were RBC-lysed, centrifuged, resuspended, and stained with a PE-conjugated anti-CD3 antibody (Cat. No. MAI-10179, Thermo Scientific) at 4°C for 30 mins and analyzed on Accuri C6 flow cytometer.

Phagocytosis (ADCP) Assay Alemtuzumab (Campath, anti-CD52, Genzyme), was pre-treated with different concentrations of imlifidase to generate F(ab’)2, scIgG alemtuzumab. Samples were run on SDS-PAGE to confirm cleavage status.

Target NuDUL-1 cells were washed in PBS and stained with calcein at RT for 10 min in the dark. Cells were washed and resuspended in RIO medium at a concentration of 1 x 10 ⁶ cells/mL. THP-1 effector (E) cells, cultured in RIO medium, were washed and resuspended in PBS before staining with FarRed. After two washes, the cells were resuspended in RIO medium (1 x 10 ⁶ cells/mL).

The NuDULl target cells were seeded in V-shaped wells (96-well plate), together with imlifidase generated F(ab’)2, scIgG, or intact alemtuzumab at a final concentration of 10 pg/mL and incubated for 20 min prior to incubation with THP-1 effector cells. A sample without alemtuzumab was also prepared as negative control.

The THP-1 effector cells (200 000/well) and target cells (10 000/well) were incubated at 37°C, 5% CO2 for 90 min. The plate was centrifuged and the supernatant discarded. Resuspended cells were fixed with 4% PFA for 3 min before being resuspended in PBS (+0.5% BSA) after removal of the supernatant, and transfer to FACS tubes for analysis using the Accuri C6 flow cytometer.

In Vivo F(ab’)2 elimination after imlifidase treatment

Fifteen healthy individuals from a Phase I Study (study number 18-HMedIdeS-15) receiving one i.v. dose of 0,25 mg/kg body weight imlifidase. Pharmacodynamic samples were collected predose and up to 144 hours post dose and analyzed using a 10 well 4-20% Mini-PROTEAN®TGX™ Stain Free gel. The serum samples were diluted 1: 10 in PBS and 10 pL diluted serum samples mixed with 30 pL 2* SDS-PAGE loading buffer. Samples were heated at 92°C for 3 min before loading 10 pL of each serum sample onto. 5 pL molecular size standard (PPP) were used. Samples were separated using l x Tris/Glycine/SDS buffer at 200 V for 40 min. The gels were activated for 45 s and scanned for 0.5-1 s using ChemiDoc Imaging system. To follow the in vivo elimination of imlifidase cleavage products, gel images from 15 individuals were evaluated using a visual scoring judging by the presence (+) or absence (-) of the F(ab’)2 fragments over time.

Results

Autologous or allogeneic cell therapies can be curative treatment options for patients with malignant (e.g. ALL, AML) and non-malignant diseases (e.g. immune deficiencies, Thalassemia). Safe and efficacious treatments need to overcome several hurdles, including the preconditioning of the host to eliminate malignant or defective hematogenic cells and to allow for the establishment of donor BMC. One of the challenges is to find treatment regimens that strikes a balance between sufficiently conditioning the host by eliminating pathogenic cells without compromising a swift reestablishment of the hematopoietic system by donor BMC. If this balance is not achieved, complications such as GvHD, graft failure (GF) and life-threatening infections may occur.

To achieve that balance, complex treatment regimens need to be adjusted for dosage and timing of e.g., irradiation and lymphodepleting drugs, like ATG or alemtuzumab. The IgG-based biologies rabbit ATG and anti-CD52 alemtuzumab bind and kill potentially both, donor- and host lymphocytes. On one hand, using high doses of these drugs leads to a desired reduction of graft-versus-host disease (GvHD), but at the cost of delayed immune reconstitution or even increased host engraftment failure, thereby enhancing the risk of severe infectious complications and overall survival (OS). Persisting levels of alemtuzumab or of ATG in the patient’s blood (and bound to endothelial cells) creates yet another level of intricacy, delaying the immune recovery after HSCT which are associated with an increased risk of severe viral reactivations.

The dilemma of needing sufficiently high doses of IgG-based biologies during preconditioning, but low levels at the time of BMT can be solved using IgG-inactivating enzymes, such as imlifidase and EndoS. For this concept to work the biologies need to be cleavable by imlifidase. Fig. 3 shows that polyclonal rabbit ATG (ATG-G or Thymoglobulin), as well as, the human IgGl -based mAb alemtuzumab are efficiently cleaved in vitro by imlifidase, into their respective 2*Fc/2 and F(ab’)2 fragments by imlifidase in buffer. Rabbit ATG and human IgGl -based alemtuzumab are thus examples of biologies that can be uncoupled from their Fc-effector functions and potentially be inactivated in vivo through imlifidase cleavage. In contrast, the horse polyclonal IgG thymoglobulin cannot be cleaved by imlifidase.

Even though the Fc-effector function of these antibodies was neutralized, it is still of interest if and at which dose their 2*Fc/2 and F(ab’)2 fragments exert cytotoxic effect on target cells. The ability to kill K562 target cells was tested as a readout system to assess NK survival and functionality. NK cells were incubated with different concentrations of intact antibody (IgG) or their respective in vitro-c\Qa.vQ 2*Fc/2 and F(ab’)2 fragments.

The cytotoxic potency of rATG-F(ab’)2 towards eNK cells was reduced compared to the equivalent concentrations of intact IgG (Fig.4). After incubation of NK cells with 100 pg/mL intact rATG only 20 % of the K562 target cells were lysed, demonstrating an impairment of the NK cells. In contrast, the equivalent dose of rATG F(ab’)2 still allowed the NK cells to lyse most of their target tumor cells. This thus demonstrates that rATG F(ab’)2 is less cytotoxic than intact rATG, even if it retains some of its cytotoxic activity at very high doses.

The protective effect of imlifidase cleavage of biologies is even more prominent when using the anti-CD52 targeting antibody alemtuzumab. A concentration of 2 pg/mL alemtuzumab efficiently inactivates the NK-effector cells, exhibiting an equivalent reduction of K562 target cell lysis at 100 pg/mL alemtuzumab. On the other hand, equivalent fragment doses of 100 pg/mL alemtuzumab, i.e. 75,3 pg/mL F(ab’)2 or 24,7 pg/mL 2*Fc/2 fragments have no negative effect on NK cells. Neither 2*Fc/2 purified from imlifidase-cleaved rATG (Fig. 4c) nor from alemtuzumab (Fig. 4f) had a blocking effects of NK cell activity.

ADCP is one of the effector mechanisms by which alemtuzumab can mediate the removal of CD52-positive target cells. Alemtuzumab was incubated with different concentrations of imlifidase to address the question which activity the different degrees of IgG digestion have on ADCP. The digests were run on SDS-PAGE to facilitate the visualization of the samples that were fully cleaved by imlifidase into F(ab’)2 and 2*Fc/2 fragments, predominantly into scIgG, or intact alemtuzumab as positive control (Fig.5a). NuDULl target cells were incubated with either intact IgG, scIgG or fully imlifidase-cleaved F(ab’)2 and 2*Fc/2 alemtuzumab fragments to test their ability to mediate ADCP by monocytic THP1 cells. At a concentration of 30 pg/mL both, single cleaved and intact alemtuzumab were able to mediate ADCP to the same degree (Fig.5b). A dose titration of the different alemtuzumab preparations shows that scIgG alemtuzumab completely loses its ability to opsonize CD52-positive NuDULl target cells at around 2 pg/mL, while intact alemtuzumab displays the same potency at 2 pg/mL and 30 pg/mL. Completely cleaved alemtuzumab into 2*Fc/2 and F(ab’)2 by imlifidase fully loses its ability to mediate ADCP, even at 30 pg/mL.

After incubation with indicated doses of intact alemtuzumab or thymoglobulin, flow cytometry analysis of whole blood shows that both drugs efficiently reduce CD3+ positive T- cells already after 4 hours incubation (Fig. 6). In a next step, whole blood was preincubated with different doses of imlifidase for 6h before adding alemtuzumab (100 pg/mL) to the preconditioned blood and incubated for another 16h. Flow cytometry shows that imlifidase at 1.9 pg/mL is able to cleave endogenous IgG (Fig.7a) and able to block the cytotoxic effect of 100 pg/mL alemtuzumab on CD3-positive T-cells (Fig.7b). In contrast, the horse IgG-based drug ATGAM is not cleavable by imlifidase (Fig. 3d), and its cytotoxic activity in whole blood is not reduced even at 30 pg/mL imlifidase (Fig. 8b), while the endogenous IgG pool is fully cleaved (Fig.8a).

The role of CDC in the early cytotoxic activity of alemtuzumab (30 pg/mL) and horse ATGAM (100 pg/mL) was shown by pre-incubating whole blood with the C5 inhibitor eculizumab. The complement cascade inhibition by eculizumab prevented most of the cytotoxicity by alemtuzumab and ATGAM. Single- and fully-cleaved alemtuzumab by imlifidase show the same degree of CDC blocking (Fig.9).

Imlifidase has not only the ability to cleave serum IgG in vivo but it also cleaves therapeutic antibodies such as rabbit ATG and alemtuzumab into 2*Fc/2 and F(ab’)2 fragments in the presence of serum matrix. The detachment of the Fc from IgG allows the F(ab’)2 fragment not only to be eliminated faster due to its reduced molecular size but also due to the lack of FcRn-recy cling, otherwise observed with intact IgG. Imlifidase-treated individuals exhibit a decline of F(ab’)2 in serum when analyzed by SDS-PAGE, showing that full cleavage of intact IgG occurs within a couple of hours, as evidenced by the vanishing of the 150 kDa IgG band. Instead, a 100 kDa F(ab’)2 and 25 kDa Fc/2 band is observed on SDS-PAGE. The intensity of the 100 kDa F(ab’)2 band disappears to a great extend within 72 hours in most imlifidase-treated individuals (n=15), exemplified for one individual (ID-2) in Fig. 10a. These in vivo experiments show that imlifidase not only inactivates Fc-effector functions but also accelerates the decline of remaining F(ab’)2 with potentially cytotoxic activity as observed for rATG.

Discussion

Cell therapies such as hematopoietic stem cell transplantation (HSCT) can be curative treatment options for patients with malignant diseases, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and for patients with non-malignant hematological diseases, e.g., immune deficiencies, Thalassemia and sickle cell disease. Preconditioning of the host is not only needed to create space for the donor HSC in the hematological niches, but also to suppress graft rejection by host immune cells, to avoid GvHD, and to eliminate the pathogenic host cells. Regimens including total body irradiation and administration of e.g. IgG-based biologies need to be fine-tuned to balance the desired with the negative effect from those treatments.

It has been shown that higher rATG doses correlate with lower BMTx rejection rates and lower incidence of acute and chronic GvHD. On the other hand, the same treatment also increases the risk of delayed immune recovery, i.e. NK and T-cell levels, rendering the patient more susceptible to life-threating infections. The recovery of the T-cell pool can exceed one year (Servais et al 2015). This problem can be solved by inactivating those IgG- based biologies prior to HSCT. The IgG-based biologies can be inactivated by imlifidase, which effectively hydrolyzes human and rabbit IgG in the lower hinge region creating one F(ab’)2 fragment and one homodimeric 2*Fc/2-fragment. The resulting F(ab’)2 fragment retains binding capacity to antigens (e.g. CD52) but their Fc-dependent effector mechanisms, such as complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are eliminated. Since the mechanisms of action of rATG (such as Grafalon and Thymoglobulin) and alemtuzumab are, to a large extent, Fc-mediated, imlifidase can be used as a tool to inactivate these drugs and limit cytotoxicity towards NK cells before HSCT. Furthermore, imlifidase is also of use in cases where some of the cytotoxic effector mechanism is mediated through the crosslinking of receptors, which would be achieved due to the accelerated elimination rate of F(ab’)2 fragments compared to intact IgG.

Alternative methods of IgG removal, like plasmapheresis or plasma exchange, are inferior, since the drug antibodies i.e. rATG or alemtuzumab cannot be removed completely from the patient due to inevitable backflow from the tissue and interstitial fluids (Achini et al., 2020; Zhang et al., 2019). The same is true for already cell-bound antibodies. Imlifidase on the other hand, will inactivate Fc functions right away in the blood compartment as well as in interstitial fluid and even of already cell bound antibodies. The fast mode of action of imlifidase might also be useful in cases where no antidote is available for an accidental overdose of IgG-based drugs (van der Zwan et al., 2018), which need to be inactivated instantaneously. Imlifidase is also superior because it can be administered within minutes, whereas plasmapheresis or plasma exchange are more strenuous for patients.

A similar problem, with a negative impact on the outcome of cell therapies after the use of therapeutic antibodies, has been seen with antibodies modifying the T cell population such as PD-1 and PD-L1 antibodies. It is well known that for instance anti-PDl (nivolumab) can affect the outcome of HSPC and it is therefore accompanied with a warning on the package insert (OPDIVO U.S. Prescribing Information (bms.com)) stating "Fatal and other serious complications can occur in patient who receive allogeneic HSCT before or after being treated with a PD-1/PD-L1 blocking antibody". The use of an IgG degrading enzyme to inactivate this type of antibodies may have a significant benefit for patients as the half-life of the antibodies when cleaved into F(ab')2 and Fc would be significantly shortened. References

- Servais S, Menten-Dedoyart C, Beguin Y, Seidel L, Gothot A, Daulne C, Willems E, Deiens L, Humblet-Baron S, Hannon M, Baron F. PLoS One. 2015 Jun 22;10(6):e0130026.

- Achini FR, Smiers F, Jan Zwaginga J, van Tol MJD, Joi -van der Zijde CM, Schilham MW, Lankester AC, Bredius RGM. Bone Marrow Transplant. 2020 Aug;55(8): 1671-1673.

- Zhang P, Curley CI, Mudie K, Nakagaki M, Hill GR, Roberts JA, Tey SK. Bone Marrow Transplant. 2019 Dec;54(12):2110-2116.

- van der Zwan M, Baan CC, van Gelder T, Hesselink DA. Clin Pharmacokinet. 2018 Feb;57(2): 191-207.

Example 3 - dosing and timing intervals for imlifidase with alemtuzumab, ATG-G and Thymoglobulin

The following scheme for each of alemtuzumab, ATG-G and Thymoglobulin will be used in a clinical setting. Cell transplant takes place at day = 0. Example 4 - Reduction of phagocytosis of opsonized platelets

IgG antibody decorated platelets may be prone to elimination by the immune system via Fc-mediated effector mechanisms including CDC, ADCP, or hypercoagulation (Cumpelik et al., 2015).

The origin of autologous platelet-specific antibodies may stem from autoimmune processes leading to immune thrombocytopenia (ITP). Alternatively, TCD -antibody-related complications are common during heterologous platelet transfusion (e.g. thrombocytopenia and platelet refractoriness after HSCT) when the recipient has undergone AB- and cell- mediated platelet destruction and/or suppression of platelet production or has previously developed donor-specific antibodies (DSA). Furthermore, platelet-specific antibodies can originate from therapeutic antibody preparations received during induction therapy regimens for e.g. hematopoietic stem cell or solid organ transplantations (Ankersmit et al, 2003). Such treatments may include polyclonal rabbit anti-thymoglobulin globulin (rATG) preparations, which have been shown to bind to platelets (Langer et al. 2006). Moreover, Alemtuzumab is associated with the development of thrombocytopenia and affecting the responsiveness to conventional ITP therapies (Cuker et al., 2011).

Besides their role as cellular mediators of thrombosis, platelets also act as immune cells that are believed to be involved in the innate immune response as well as playing a role in acquired immune responses. Their depletion can thus have a detrimental effect on a patient’s immune response and risk to hemorrhage

To protect platelets from adverse effects caused by antibodies, imlifidase could reduce IgG mediated effector mechanisms.

Materials and methods

Preparation of leukocyte pure - Platelet Rich Plasma (P-PRP)

Platelet rich plasma (PRP) samples (n=5) were collected in 0.32% Na-citrate (BioIVT, #HUMANPL32NC, UK and delivered on cold packs. Upon arrival plasma was processed as follows to generate leukocyte pure - PRP. Remaining RBC were removed from PRP by 400 g for 10 min centrifugation step. The clear PRP supernatant was collected and centrifuged at 800 g for 10 min to pellet remaining leukocytes. The upper plasma fraction was collected (P-PRP) and stored at 4-8°C until use ADCP assay.

Staining of Platelets target cells and THP-1 effector cells Plasma proteins were removed from platelets (Pits) before Calcein-AM (Invitrogen, # C3099) staining as follows: P-PRP were washed with Na-Citrate buffer 0,38% (pH 7) to remove serum proteins before Calcein-AM staining as follows: P-PRP were diluted with Na- Citrate and centrifuged at 1600 g for 5 min. Supernatant was carefully removed to not disturb the pelleted Pits. Pits were resuspended in Na-Citrate buffer and spun at 1600 g for 5 min. Supernatant was removed and the pelleted Pits resuspended with their original plasma volume with Na-Citrate buffer. After plasma protein removal the platelets were stained with Calcein— AM for 15 min. The staining reaction was quenched by washing with Na-Citrate supplemented with 1% BSA followed by centrifugation at 1600 g, 5min.

Cell culture medium proteins were removed from monoblastic THP-1 effector cells (ACC16, DSMZ, Germany) using PBS-D as washing buffer (2x at 400 g) before staining with FarRed DDAO-SE (Molecular Probes, # C34553). The staining reaction was quenched by washing with DMEM 1% BSA.

ADCP assay

Pit target cells were incubated with or without imlifidase treated Grafalon 100 pg /ml or Thymoglobulin 200 pg /ml final cone, before adding THP-1 effector cells. Assay plates were incubated for 90 min at 37°C.

Cells were analysed using the Beckman Cytoflex flow cytometer platform in FITC (488/520 nm) for Calcein and APC (670/30 nm) for FarRed to identify phagocytosed Pits in the Calcein-AM/FarRed double positive population.

Statistics

A paired T test (GrapPad Prism) was used to calculate significance.

Results

Calcein stained Pure-Platelet Rich Plasma (P-PRP) cells from five donors were opsonized with and without imlifidase-treated Grafalon (100 pg /ml) or Thymoglobulin (200 pg /ml) before adding phagocytic FarRed labeled THP1 effector cells. After 90 min incubation the cells were analysed by flow cytometry for Calcein and FarRed expression as shown in dot plot (Fig. I la). All Calcein positive populations were further divided into “Calcein + / FarRed-” (platelets only) and “Calcein + / FarRed +” double positive cells (phagocytosed platelets) as shown (Fig.1 lb). The calculated percentage of the Calcein/FarRed double positive is based on all Calcein positive cells set as 100%. Changes in phagocytosis with and without imlifidase treatment is shown for Grafalon (Fig.l 1c) and Thymoglobulin (Fig.1 Id) for five individuals. Imlifidase treatment of both rabbit ATGs leads to significant reduction in phagocytosis (paired T test analysis, p < 0.05).

Using an in vitro ADCP assay, the inventors have shown that the IgG degrading enzyme imlifidase can reduce phagocytosis of Grafalon and Thymoglobulin opsonized platelets (see Fig. 11).

References

- Cumpelik el al. Am J Transplant. 2015 Oct; 15(10):2588-601.

- Ankersmit et al., ' Am J Transplant. 2003 Jun;3(6):754-9.

- Langer et al. Blood (2006) 108(11); 3905.

- Cuker et a/. Blood. 2011 Dec 8; 118(24):6299-305. doi: 10.1182/blood-2011 -OS- 371138. Epub 2011 Sep 29. PMID: 21960587.

Example 5 - Modification of T cell-depleting agents by imlifidase

The inventors have discovered that administration of imlifidase after TCD-Ab treatment and before graft infusion counterbalances the negative effect of functional TCD-Ab agents on T and NK cells in recipients of hematopoietic stem cells. To confirm this, the inventors have shown that imlifidase in vitro, by cleaving TCD-Abs in F(ab’)2 and Fc fragments, i.) reduces the rates of apoptosis and necrosis of T and NK cells, ii.) efficiently preserves the function of T and NK cells in comparison to T and NK cells exposed to uncleaved Grafalon, Alemtuzumab, or Thymoglobulin, respectively.

Materials and methods

Expanded NK cells (eNKs) were generated by incubating isolated PBMCs from healthy donors with IL-2 (50-100 U/ml, Novartis) for 14 days in the presence of irradiated feeder cells (feeder cell line: K562-mbIL15-4-lBBL).

If not freshly used in subsequent assays, eNK cells were frozen. Before use in assays, frozen eNKs were thawed and cultivated overnight (cell density: 3xl0e6/ml) in RPMI 1640 medium (Gibco) supplemented with IL-2 (100 U/ml) and heat-inactivated human AB serum (10%, Bio & Sell).

Calcein release assays Calcein release assays were performed to test NK cell-mediated cytotoxicity against K562 cells, preceded by two preincubation periods (preincubation I and II), as shown in Figure 1. Two independent experiments were performed.

During preincubation I,Grafalon, Thymoglobulin, Alemtuzumab, or control-Abs (rlgG / hlgGl), respectively, were incubated with or without imlifidase, 10 pg/ml for 1.5 h. TCD-Abs precleaved with imlifidase are referred to as “fragment mix”.

During preincubation II, freshly expanded or thawed eNK cells, resuspended in RPMI 1640 medium containing functional human AB serum (final concentration: 25%), were thereafter added to the TCD-Abs or the “fragment mix” with an incubation time of 3.5 h. As controls, effector cells were either added to the fragment mix of rlgG / hlgGl or to full length rlgG / hlgGl .

Prelabeled K562 cells were used as target cells in the subsequent calcein release assay with an incubation time of 2 h. The amount of released calcein which parallels NK cell-mediated cytotoxicity was measured fluorometrically. Lysis (%) was calculated as follows:

[(test release minus spontaneous release) / (maximum release minus spontaneous release)] * 100.

Spontaneous release represents the release of calcein from target cells in medium alone, maximum release the calcein release from target cells lysed in medium plus 1% Triton X-100 (each measured in triplicates). Spontaneous release (%) was calculated as follows:

[(spontaneous release minus background) / (maximum release minus background)] * 100.

Background: medium plus supernatant of calcein-labeled cells. In all figures displaying NK cell cytotoxicity assays based on calcein release detection, means of triplicates with SD are shown.

Mean lyse rates of E+T of each donor and each assay in the absence of the respective TCD-Ab were set to 100% in order to allow comparisons between donors / assays. Accordingly, lyse rates in the following paragraphs are “normalized” lyse rates. If not otherwise indicated, the following parameters were kept constant: E:T ratio = 10: 1; concentration of imlifidase 10 pg/ml; functional (=non-heat-inactivated) human AB serum 25%; duration of preincubation I (preincubation II, calcein release assay): 1.5 h (3.5 h, 2 h). Viability assays Apoptosis was assessed with an Annexin V-FITC staining kit (Miltenyi Biotech, Germany). eFluor 780 (Invitrogen, MA, USA) was used as a marker for necrosis. Two independent experiments were performed.

PBMCs from healthy donors were isolated using Ficoll density centrifugation, then resuspended in RPMI 1640 medium supplemented with functional human AB serum (final concentration: 25%) and plated on 96 well plates (cell density: 2x 10e6/ml). PBMCs were thereafter incubated (for 2 h, if not otherwise specified) with: i.) ATG-G (Grafalon), Alemtuzumab (Lemtrada), or Thymoglobulin or ii.) the corresponding fragment mixes of ATG-G, Alemtuzumab, or Thymoglobulin (fragment mixes were generated in advance by incubating full length ATG-G, Alemtuzumab, or Thymoglobulin with imlifidase for at least 1.5 h)

As controls, PBMCs from the same donors were incubated (for 2 h, if not otherwise specified), in parallel, with: i.) rlgG or hlgGl or ii.) the corresponding fragment mixes of rlgG or hlgGl (fragment mixes were generated in advance by incubating full length rlgG or hlgGl with imlifidase for a minimum of 1.5 h);

After two washing steps utilizing lx binding buffer (Miltenyi Biotech, Germany), FACS staining was performed using antibodies to detect CD3, CD56 and CD14. Annexin V and eFluor780 were added to analyze viability (early / late apoptosis; necrosis) of PBMCs. FACS analysis was thereafter performed on a FACS Canto II instrument using FACSDiva™ software (both BD Biosciences, CA, USA). Acquired data were further analyzed with FlowJo software version 10.8.0 (Tree Star Inc., OR, USA) and GraphPad Prism, version 9.4.1 (GraphPad Software Inc., CA, USA).

Results

Mean lyse rates of E+T of each donor / each assay in the absence of TCD-Abs were set to 100% in order to allow comparisons between donors / assays. Accordingly, lyse rates in the following paragraphs are “normalized” lyse rates.

Irrespective if ATG-G (Grafalon), Alemtuzumab, or Thymoglobulin were used, “targets only” were not lysed. This indicates that lysis of K562 cells, as shown in the calcein release assays, is not due to a direct effect of the TCD-Abs solely, but rather requires the presence of eNKs as effector cells. Production and expansion of effector cells

Figure 13 demonstrates the flow cytometric results of eNKs of one representative NK cell donor. Expanded cells consist of ~ 90% CD56 ⁺ NK cells with significant co-expression of CD 16 and of CD69 (activation marker).

Influence of ATG-G (Grafalon) on NK cell-mediated lysis

ATG-G reduced NK cell-mediated lysis dose-dependently in all donors in the first experiment (Figure 14a, ATG-G is referred to as ATG in the figure). Also, target cell lysis was efficiently enhanced in all donors when TCD-Abs had been precleaved by imlifidase. In the presence of 10 pg/ml (100 pg/ml) ATG-G, lyse rates ranged from 35% - 60% (0% - 15%), whereas lyse rates rose up to 70% - 85% (40% - 65%) in the presence of the corresponding ATG-G fragment mixes.

The second experiment confirmed these results; ATG-G (Grafalon) reduced NK cell- mediated lysis dose-dependently in all donors (Figure 14b). In the presence of full length ATG-G, depending on NK cell donor, lyse rates ranged from 61% to 88% (ATG-G 10 pg/ml) and from 33% to 40% (ATG 100 pg/ml). With the use of imlifidase (10 pg/ml), i.e. in the presence of fragment mixes of ATG-G, effector cell lysis was significantly enhanced in all three donors. This effect of imlifidase was more profound the higher the concentration of ATG-G was. Compared to lysis in the presence of uncleaved ATG-G 100 pg/ml (10 pg/ml), lysis was enhanced by 37% to 60% in the presence of the ‘fragment mix of ATG-G 100’, but by only 8% to 45% in the presence of the ‘fragment mix of ATG-G 10’.

With respect to the polyclonal rabbit control Ab preparation (rlgG), rabbit IgG (100 pg/ml) was also able to reduce effector cell lysis and imlifidase-cleaved rlgG (10 pg/ml) was also able to enhance lysis in this context. The effect of the non-specific rlgG is hypothesized to be due to IgG aggregates, which trigger fratricide of NK effector cells via FcgR crosslinking. Imlifidase treatment of rlgG would thereby break the crosslinking by removing the Fc parts of the IgG aggregates.

Influence of Alemtuzumab on NK cell-mediated lysis

In the presence of alemtuzumab, NK cell-mediated lysis of effector cells was significantly reduced in the first experiment (Figure 15a). However, in contrast to ATG-G, a dose-dependent effect on NK cell lysis was not observed. The effectiveness of alemtuzumab was still in plateau at concentrations between 0.5 - 100 pg/ml. In the presence of 1 pg/ml (100 pg/ml) Alemtuzumab, lyse rates ranged from 30% - 60% (38% - 65%), while in the presence of the corresponding fragment mixes of Alemtuzumab, effector cell lysis was markedly enhanced and reached rates > 90%. The second experiment confirmed these results. With the use of Alemtuzumab, NK cell lysis was significantly reduced in all donors (Figure 15b). Again, contrary to ATG-G, this impact of alemtuzumab on NK cell function was not dose-dependent in the tested range (1 - 100 pg/ml). Using full length Alemtuzumab with 1 (10; 100) pg/ml, lyse rates ranged between 29% - 70% (38% - 72%; 54% - 80%).

Using imlifidase (10 pg/ml), lyse rates were drastically enhanced in all donors, even at high concentrations of alemtuzumab (100 pg/ml).

Influence o f Thymoglobulin on NK cell-mediated lysis

Higher doses of Thymoglobulin (200 pg/ml) reduced NK cell-mediated lysis of effector cells stronger than 1 pg/ml of Thymoglobulin in all donors in the first experiment (Figure 16a). Using imlifidase, lysis was increased in all donors at low as well as at high concentrations of Thymoglobulin. In the presence of 1 pg/ml (200 pg/ml) Thymoglobulin, lyse rates were between 30% - 90% (5% - 30%) and rose up to > 95% (30% - 80%) in the presence of the respective Thymoglobulin fragment mixes.

Results from the second experiment confirmed these results. Using Thymoglobulin (full length), NK cell-mediated lysis was reduced in a dose-dependent manner in all donors (Figure 16b). Depending on NK cell donor, lyse rates ranged from 78% - 93% after preincubation of eNK cells with 10 pg/ml Thymoglobulin, but were further reduced to 32% - 54% after exposure of eNK cells to 200 pg/ml Thymoglobulin. In general, NK cell function was increased in all donors with the use of imlifidase (10 pg/ml). This imlifidase-derived effect was more obvious at higher doses of Thymoglobulin (200 pg/ml). Fragment mixes of Thymoglobulin 200 efficiently increased lysis by up to 43% (range: 19% - 43%) as compared to the lyse rates in the presence of uncleaved Thymoglobulin (200 pg/ml).

Effect of ATG-G (Grafalon) on T and NK cell viability

To assess the effect of TCD-Abs on necrosis and induction of apoptosis in T and NK cells, Annexin V / eFluor 780 assays were performed.

In the first experiment, in the absence of ATG-G, 92% of T cells (mean of 4 donors) remained viable after 2 h. Viability was neither decreased by rlgG nor by imlifidase itself. In the presence of ATG-G, viable T cells were dose-dependently reduced, apoptotic T cells, in parallel, increased; At 10 (100, 1000) pg/ml ATG-G, 76% (29%, 11%) of T cells remained viable, whereas 17% (47%, 66%) of T cells became (early) apoptotic (Figure 17a, ATG-G is referred to as ATG in the figure). However, in the presence of the corresponding concentrations of ATG-G “fragment mixes”, viability significantly increased to 94% (81%, 44%). Thus, imlifidase was able to efficiently reduce ATG-G’s impact on T cell viability, even at higher ATG-G concentrations (ATG 100).

In the second experiment, when ATG-G was absent, 92% (mean of 4 different donors) of T cells remained viable after an incubation period of 2 h. Neither rabbit IgG (rlgG) control antibody (100 pg/ml), nor ‘imlifidase (10 pg/ml) solely’ decreased T cell viability in the absence of ATG-G (Figure 17b).

In the presence of ATG-G, the amount of viable T cells was again dose-dependently (ATG-G 10, 100, 250 pg/ml) reduced, whereas (early / late) apoptotic T cells increased: at a concentration of 10 (100, 250) pg/ml of uncleaved ATG-G, 86% (51%, 37%) of T cells remained viable, whereas 13/1,4% (38/11%, 48/15%) of T cells became (early / late) apoptotic.

Similar to the first experiment, viability of T cells was relevantly increased when ATG-G had been precleaved by imlifidase; In the presence of the ‘fragment mix of ATG-G 100 (250)’, viability rose up from 51% to 77% (from 37% to 72%).

Regarding NK cells (Figure 17c), counts of viable cells were increased in the first experiment when ATG-G had been preincubated with imlifidase from 73% to 96% (ATG-G 10 vs. ATG- G 10 + IdeS 10), from 37% to 94% (ATG-G 100 vs. ATG-G 100 + IdeS 10), from 15% to 60% (ATG-G 1000 vs. ATG-G 1000 + IdeS 10).

In the second experiment, viable cells were similarly lowered in a dose-dependent manner, (Figure 17d), ranging from 91% in the absence of ATG-G to 65% (34%, 24%) in the presence of 10 (100, 250) pg/ml ATG-G. In the presence of the corresponding concentrations of ATG-G fragment mixes, viability of NK cells was again significantly increased to 91% (95%, 91%).

Effect of Alemtuzumab on T andNK cell viability

Alemtuzumab decreased the viability of both T cells (Figure 18a) and NK cells (Figure 18c) in a dose-dependent manner in the first experiment. Without Alemtuzumab, mean viability of T (NK) cells was 94% (96%) after 2 h. In the presence of 1 (10, 100) pg/ml Alemtuzumab, viability of T cells was reduced to 82% (11%, 12%), and viability of NK cells to 87% (67%, 54%). Thus, viability of NK cells seems to be less affected by Alemtuzumab than the viability of T cells. Even so, viability of T cells preincubated with either Alemtuzumab 100 + IdeS 10 or Alemtuzumab 10 + IdeS 10 was as high as in the absence of Alemtuzumab. The same result was observed in the case of NK cells.

Altogether, this indicates that F(ab’)2 fragments of Alemtuzumab, even if present at higher concentrations (Alemtuzumab 100 + IdeS 10), do not seem to compromise T and NK cell viability. This finding is surprising as some F(ab’)2 fragments are known to induce cytotoxicity, but the F(ab’ fragments stemming from imlifidase-cleaved Alemtuzumab do not have this effect.

Results from the second experiment confirmed these results; Alemtuzumab decreased T (Figure 18b) and NK cell (Figure 18d) viability in a dose-dependent manner (1, 10, 100 pg/ml). However, viability of NK cells was less affected than T cell viability (10, 100 pg/ml Alemtuzumab). In the absence of Alemtuzumab, mean viability of T (NK) cells was 88% (92%) after 2 h. After a two-hour incubation with 1 (10, 100) pg/ml Alemtuzumab, viability of T cells was reduced to 78% (20%, 14%), whereas viability of NK cells only to 75% (58%, 51%).

Viability of T cells which had been preincubated with ‘fragment mixes of Alemtuzumab 1 (10, 100)’ remained as high as in the absence of Alemtuzumab (Figure 7b. The same result could be observed in the case of NK cells (Figure 18d).

Effect of Thymoglobulin on T and NK cell viability

In the first experiment, T and NK cell viability was only slightly affected by 1 and 10 pg/ml of Thymoglobulin. However, viability of both cell types was markedly reduced by higher doses of Thymoglobulin. In the presence of 100 pg/ml (200 pg/ml) of Thymoglobulin, only 37% (23%) of T cells and only 23% (12%) of NK cells remained viable (Figure 19a and c). With the use of imlifidase to precleave Thymoglobulin, NK cell viability (89% - 96%) was nearly as high as in controls, and T cell viability was increased to 74% - 93%.

Results from the second experiment confirmed these results. Both T and NK cell viability were markedly reduced by high concentrations of Thymoglobulin: with 200 pg/ml Thymoglobulin, only 38% of T cells and NK cells remained viable (Figure 19b and d). Since T cell viability was only slightly decreased after a two-hour incubation with 2 or 10 pg/ml Thymoglobulin (e.g. reduction of T cell viability by only 7% by 10 pg/ml Thymoglobulin), the effect of imlifidase in enhancing T cell viability through pre-cleavage of Thymoglobulin in this setup (incubation time: 2 h) was therefore limited (Figure 19b). However, in comparison, T cell viability at 200 pg/ml Thymoglobulin (38%) was increased considerably in the presence of ‘fragment mix of Thymoglobulin 200’ (70%).

In contrast to T cell viability, viability of NK cells could be rescued even more efficiently by imlifidase: even at high concentrations of Thymoglobulin (200 pg/ml), NK cell viability was increased from 38% to 93%, thus, reaching levels as high as NK cells in the absence of Thymoglobulin (Figure 19d). In parallel to ATG-G, NK cell viability was enhanced to the by ‘fragment mix of Thymoglobulin’. Discussion

TCD-Abs are essential drugs to prevent graft rejection. However, they display adverse effects on post-transplant immune recovery - an ongoing relevant problem in transplant medicine. Ideally, TCD-Abs should be inactivated by imlifidase before graft infusion, thereby preserving the function of co-transfused immune effector cells without enhancing the risk for graft rejection.

Indeed, it was shown that by pre-treatment of all three TCD-Abs with imlifidase, viability of NK and T cells was markedly improved as compared to NK and T cells exposed to uncleaved TCD-Abs. Furthermore, imlifidase efficiently preserved the function of eNK cells after cleaving clinically relevant doses of Grafalon, Alemtuzumab, and Thymoglobulin. These results suggest that the methods of the invention allow for the use of high doses of TCD antibodies for preconditioning, while sparing otherwise negatively affected cells important for immune recovery.

Example 6 - Improved CAR T-cell survival after inactivation of preconditioning and lymphodepleting biologies by imlifidase

Patients with hematopoietic cancers are not only treated with IgG-based biologies like rituximab, alemtuzumab or rATG during first and second line treatments but also during bridging therapies (Bhaskar-2021) for HSC transplantation, or for the debulking of the tumor burden prior to CAR cell therapy infusions. Lowering the tumor burden through preconditioning can reduce the risk for CAR therapy-induced tumor lysis syndrome.

Furthermore, the use of biologies and other lymphodepleting measures prevents the kill ing of infused CAR cells by the host T-cells, which might be desired especially a problem after repeated dosing with allogeneic CAR cells.

An additional reason to use lymphodepleting agents can be to open a niche for the expansion of the CAR cells. Lymphodepleting conditi oning can therefore even be beneficial before the 1st infusion of autologous CAR cells.

Despite the benefits of lymphodepletion there are also drawbacks when combined with CAR T-cell treatments. CD52 positive CAR cells are vulnerable for depletion by remaining anti- CD52 antibodies like alemtuzumab. This problem has been recognized and prompted companies to develop CD52-deleted CAR cells. The inactivation or deletion of CAR cells may also occur using biologies like anti-CD38, rabbit ATG such as Grafalon or Thymoglobulin, which bind to naturally expressed cell surface antigens on CAR T-cells. Furthermore, previously infused disease-related therapeutic IgGs (e.g. rituximab, cetuximab) could trigger genetically introduced antigens like a rituximab-binding RQR8 safety switches on CAR T-cells. It might be desirable to shorten the time gap between the last rituximab injection and CAR T-cell infusion, allowing for a smoother transition from e.g. debulking CD20 positive tumor cells with RTX with an effective CAR treatment.

To solve this problem, imlifidase or other IgG cleaving enzymes, can be injected to shorten the PK of biologies thus all owing the use of rituximab as a bridging therapy or to debulk big tumor burdens followed by a closely timed treatment with autologous or allogeneic safety switch CAR T-cells.

Lymphodepletion of the patient might be required for the persistence of allogeneic CAR T-cells. Therapeutic antibodies like anti-CD52 can induce a profound and lasting lympho-depletion thereby improving the persistence of allogeneic CAR T-cells. This treatment can come at the price of delayed immune recovery, which can make the patient even more vulnerable for bacterial, viral, and fungal infections. This problem is seen at high antibody doses (Smirnov-2021), probably due to the long biological half-life of these antibodies.

Reduced in vivo exposure of lymphodepleting antibodies like anti-CD52 through the degradation of enzymes like imlifidase potentially accelerates immune recovery (Owens- 2020). The inactivation of e.g., anti-CD52 by imlifidase can be of advantage for CD52- knockout and CD52-positive CAR T-cells.

Materials and methods

PBMC isolation

Leukocyte filters from whole blood in-line filters bags (CompoFlow, Cat. # CQ31450, Fresenius Kabi) were obtained from MSC Transfusionsmedicin, Vaxjd, Sweden, containing citrate phosphate dextrose (CPD) as anti-coagulant. The leukocyte filters were backflushed with D-PBS-EDTA elution buffer (D-PBS, 5 mM EDTA, 2.5% Sucrose). Cells were collected into 50 ml falcon tubes and centrifuged at 700 x g for 20 min to remove plasma. Pellets were washed 2x with D-PBS-EDTA at 700 x g for 10 min and resuspended in 20 ml D-PBS-EDTA to be layered onto 12 mL Ficoll-Paque Plus (GE Healthcare, Cat. # 17-1440- 02). Tubes were spun at 1200 x g, 20 min without breaks. PBMC band was transferred to 50 ml tubes and washed 2x with D-PBS (Gibco, Cat. # 14190-094) and spun at 700 x g for 10 min. PBMCs were aliquoted and stored in cell freezing medium (Gibco, Cat. # 12648-010) in liquid nitrogen until further use. Preconditioning CPC experiment

PBMCs were incubated (2 h, 37 °C) with T-cell depleting (TCD) antibodies such as Grafalon (Neovii Biotech GmbH) or Thymoglobulin (Genzyme), or negative control rabbit IgG (Jackson ImmunoResearch, Cat. # 011-000-002), together with baby rabbit complement (BRC) (Cedarlane, Cat. # CL3441-R). Treatment of PBMCs was followed by incubation with or without 15 pg/mL imlifidase (1 h, 37°C) to cleave antibodies. Anti-CD19 Jurkat CAR T- cells (CAR- J) (Creative Biolabs, Cat. # CARJ-ZP005) supplemented with additional BRC were added to the preconditioning mixture and incubated for 2 h at 37°C. Cells were centrifuged and washed with FACS buffer (D-PBS with 0.2% BSA) prior to preparation for flow cytometry. A schematic of the treatment regimen is shown in Figure 21.

Flow cytometry

CAR-J cells were pre-stained with 0.01 pM CellTrace CFSE (Invitrogen, Cat. # C34570) in PBS to distinguish CAR-J from PBMCs. Post-preconditioning CDC experiment, sample wells containing PBMCs and CAR-J were stained with a master mix of 7- Aminoactinomycin D (7AAD) (Invitrogen, Cat. # Al 310) live-dead stain and anti-hCD3 APC (Biolegend, Cat. # 344812). CAR-J were defined as the CFSE+ / CD3+ population and PBMC T-cells as the CFSE- / CD3+ population. Dead T-cells were defined as 7AAD+ cells in their respective populations. The percentage of dead cells was calculated based on the total gated CAR-J or PBMC T-cell population, respectively. Samples were acquired using a Beckman Coulter CytoFLEX flow cytometer and analysis was performed with Kaluza Analysis 2.1 software.

Results

The in vivo inactivation of therapeutic antibodies could be an advantage for enabling or improving next line cancer treatments in patients, e.g. receiving HSCT or CAR cell therapies. The initiation or success of cell therapies might depend on residual levels of previously infused IgG-based lymphodepleting biologies. The following experiments set out to mimic an in vitro scenario in which patients are treated to achieve profound lymphodepletion, followed by anti-CD19 CAR cell therapy. Initially (step I), it could be shown that treatment with rATG (Grafalon, 20 pg/mL) or Thymoglobulin (40 pg/mL) in the presence of complement killed more than 80% of PBMC T-cells during the preconditioning step, while negative control rabbit IgG (40 pg/mL) or PBS did not increase PBMC T-cell death (Fig. 20a).

In step II, the PBMC cell culture wells were incubated with either imlifidase (15 pg/mL) or PBS as control, before adding the anti-CD19 CAR-J T-cells, resulting in final antibody concentrations of 10 or 20 pg/mThymoglobulin, respectively, during incubation of the CAR- J T-cells.

The killing of CAR-J cells in the PBS-treated group (see Fig. 20b, black bars) reflects the remaining cytotoxic activity and Thymoglobulin after the preconditioning in step I. The imlifidase-treated group (see Fig. 20b, striped bars) improves the survival of CAR-J T-cells profoundly. These experiments demonstrate that the survival of CAR-J cells in the presence of otherwise cytotoxic levels of T-cell-depleting antibodies can be improved through administration of IgG-degrading enzymes like imlifidase. The in vivo inactivation of therapeutic antibodies, directed against naturally expressed CAR cell surface antigens, may protect CAR T-cells. Furthermore, the in vivo cleavage of therapeutic antibodies with IgG- degrading enzymes could accelerate the clearance of therapeutic antibodies, i.e. their F(ab’)2 fragments. This could facilitate the safe and effective infusion of CAR T-cells which are modified to express recombinant epitopes, like in the case of the RQR8 safety switch and rituximab.

References

- Smirnov el al. 2021 Dec, Strategies to Circumvent the Side-Effects of Immunotherapy Using Allogeneic CAR-T Cells and Boost Its Efficacy: Results of Recent Clinical Trials. Front Immunol.;12:780145

- Owens and Bozic; Bull Math Biol. 2021 Mar 19;83(5):42.

- Bhaskar et al. EJHaem. 2021 Nov 19;3(Suppl l):39-45