Siplizumab

Organ transplantation is a medical procedure in which an organ is removed from one individual, living or deceased, and placed in the body of a recipient, to replace a failing or missing organ. The different forms of organ transplantations are kidney, liver, heart, lung, pancreas, islet and small bowel.

All types of clinical transplantations require life-long immunosuppression. The currently available immunosuppressive drugs do not completely prevent immune injury to a transplanted organ, leading to suboptimal long-term results, while also carrying a substantial risk for severe side-effects such as cancer, infections, cardiovascular disease, diabetes and other metabolic derangements.

Rejection is the destructive immunological response directed towards the transplant, which will lead to organ failure unless it is prevented. The Siplizumab-based treatment developed by ITBMed is the first treatment that has the potential to give transplanted patients a life completely free from chronic immunosuppression.

Key summary

Treatment with the anti-CD2 monoclonal antibody Siplizumab has the potential to provide substantial improvements in the treatment of transplant patients, evident by the following:

  • Organ transplant patients treated with Siplizumab peri-transplant, as part of a protocol to allow complete immunosuppression withdrawal long-term, experience less chronic rejection, increased chances of being free of chronic immunosuppression, less cancer, less opportunistic infections, less treatment-related side-effects and improved quality of life.
  • Organ transplant patients treated with peri-transplant anti-CD2, specifically the parent monoclonal CD2 antibody from which Siplizumab has been humanized, experience less acute rejection compared to standard-of-care alone.
  • Organ transplant patients experiencing acute rejection can be effectively treated with anti-CD2 to reverse the rejection episode.
  • In a primate model of organ transplantation, homologous anti-CD2 monoclonal antibodies can prevent rejection, allow withdrawal of chronic immunosuppression and prolong survival.
  • The unique mechanisms of action of Siplizumab permit allospecific hyporesponsiveness, evident by the inhibition of both primary and secondary mixed lymphocyte reactions without comprising the response to non-specific stimulation.

Mechanistic rationale

Why is targeting the CD2 molecule transformative in transplantation?

ITBMed is developing a monoclonal antibody directed towards the human CD2 molecule, a 50 kDa transmembrane glycoprotein, that has a central role particularly in T lymphocyte function. The CD2 molecule is composed of two extracellular domains on which there are three major immunogenic regions (Jones E, 1992).  In humans, CD2 is expressed on all mature T lymphocytes, 95% of thymocytes and the majority of NK cells. It functions as either an adhesion or a co-stimulation molecule (Moingeon et al, 1989; Huet et al, 1986). The interaction between the CD2 and its ligand CD58 (LFA-3) mediates adhesion between T cells and antigen-presenting cells, thereby enhancing antigen recognition via the CD3-antigen-MHC receptor (Bockenstedt L et al, 1988). The CD2 molecule is also the target for an activating signal and T cell activation that can be induced in the absence of accessory cells by co-stimulation with anti-CD2 and anti-CD3 mAb (Yang et al, 1986).

Unless T cell activation is controlled, transplant rejection will be initiated. Thus, there is a clear immunologic rationale why a blocking anti-CD2 mAb can function as an immunomodulatory agent in vivo in organ transplantation. The CD2 molecule has multiple binding sites, and our highly specific monoclonal antibody Siplizumab uniquely targets a specific part of the CD2 antigen (Damschroder et al, 2004), inhibits T cell proliferation and is able to induce alloantigen-specific hyporesponsiveness (Latinne et al, 1996). This makes it an ideal drug candidate for immune regulation in organ transplantation, as the pathophysiology is based on reactions towards alloantigens.

Figure 1. Siplizumab binds to the human CD2 receptor found on T lymphocytes (T cells), natural killer cells, and thymocytes. The activation of T cells (which can harm a transplanted organ) rely on both the TCR-MHC interaction and the accessory co-stimulation of CD2/LFA-3. Studies have shown that Siplizumab has immunomodulatory effects by inducing alloantigen hyporesponsiveness, deletion of T- and NK-cells, and down-regulating the rejection response. This combination of properties have led Siplizumab to be considered an ideal drug in the treatment of transplant patients.

Siplizumab’s in vitro effects

Why is it important to detail Siplizumab´s mode of action?

This information provides a strong rationale for using Siplizumab clinically in the intended indications. The specificity of T cell activation resides in the T cell receptor/CD3 complex and is affected by accessory molecules such as CD2, which is essential for complete T cell activation to occur. These fine-tuned interactions and subsequent effects on T cell activation can be studied in detail using in vitro cellular assays. The selective deletion of activated antigen-specific T cells by addition of Siplizumab has been clearly demonstrated in vitro (Branco et al, 1999). Importantly, Siplizumab mediates deletion of activated T cells. A third unique mechanism, highly relevant for transplantation purposes, has emerged and is part of ITBMed´s proprietary research and development program.

In vitro investigation shows that Siplizumab has a high affinity for human CD2 and is capable of inhibiting a mixed lymphocyte reaction (MLR). Immunodepletion of NK cells prior to Siplizumab treatment of the MLR returned proliferative responses to control levels, with concurrent preservation of T cells. Addition of fresh autologous NK cells to NK-depleted responder populations restored Siplizumab mediated deletion activity to levels measured in the original MLR. Fixation of NK cells prior to addition to the MLR abrogated the deletion effect mediated by Siplizumab. Since the Fab fragments of Siplizumab are unable to inhibit the MLR, the mechanism of action requires an intact antibody molecule containing the Fc fragment. The effect of adding Siplizumab to MLR cultures results in a T cell population that is unable to respond to alloantigens in a secondary MLR with the same stimulators, but are still able to respond to non-specific stimulation.

“This forms the mechanistic base for using Siplizumab as a selective regulator of the immune response toward a transplanted organ, potentially achievable without affecting protective immunity towards infections and cancerous cells”  

/ David Berglund, CSO

Figure 2. Siplizumab added to a mixed lymphocyte reaction (MLR) in different concentrations. The Fab fragments of Siplizumab (F(ab’)2) as well as isotype controls (Hu IgG1) and their corresponding Fab fragments (Hu F(ab’)2) were used as controls. The MLR was continued for 6 days after which radioactive thymidine (3H-thy) was added to measure T cell activity (cpm, counts per minute). The T cell proliferation in set-up 1, 2 and 3 of the Siplizumab group was clearly inhibited (p < 0.05), demonstrating a level of proliferation at approximately 25% of the controls.

Abbreviations: 1 = 5 ug/mL, 2 = 500 ng/mL, 3 = 50 ng/mL, 4 = 5 ng/mL, 5 = 500 pg/mL, 6 = no antibodies, 7 = 5 ug/mL IgG1 isotype control, 8 = 500 pg/mL IgG1 istotype control, 9 = 5 ug/mL of Fab fragments, 10 = 500 pg/mL of Fab fragments, 11 = responder cells alone, 12 = stimulator cells alone.

Adapted from Branco et al, 1999.

Siplizumab is the humanized anti-CD2 biologic optimized from its parent antibody BTI-322

The rat monoclonal antibody BTI-322 is directed towards human CD2 and is the precursor antibody of Siplizumab. BTI-322 was humanized to form Siplizumab by exchanging as many as possible of the rat framework residues of the variable regions of the heavy and light chains for human ones, without affecting the antibody’s specificity. In addition, rat C kappa and C gamma 2b were exchanged for the human constant regions C kappa and C gamma 1, respectively. The activity of Siplizumab was compared to BTI-322 using binding assays, inhibition of the primary mixed lymphocyte reaction (MLR), and induction of hyporesponsiveness to alloantigen in a secondary MLR. The in vitro functional properties of Siplizumab were similar to those of BTI-322.

The importance of animal models

Pharmacodynamic and pharmacokinetic study in chimpanzees

Siplizumab is a humanized monoclonal antibody directed towards human CD2. The only animal species where Siplizumab itself has the same reactivity with cells as in humans is chimpanzees (Damschroder et al 2004). An in vivo comparison between BTI-322 and Siplizumab has been performed in chimpanzees, where they display similar pharmacodynamic properties, and Siplizumab is as expected less immunogenic.

In anti-CD2 treated animals, the lymphocytes were depressed, with a depth and duration lower than in the vehicle control (normal saline) treated chimpanzees (Figure 3). Lymph node biopsies done prior to and after receiving Siplizumab indicated mild to moderate depletion of lymphocytes compared to no visible change in lymph node architecture in the saline-treated chimpanzees. Lymphocyte depletion in lymph nodes lasted approximately 6 weeks, and normalized thereafter. Siplizumab concentrations obtained from the chimpanzees were judged to be consistent with a 40- to 50-hour half-life.

Targeting the anti-CD2 pathway is crucial for tolerance induction in primates

Siplizumab itself cannot be assessed directly in other animal models of organ transplantation. Instead, equivalent antibodies targeting CD2 of the respective animal model must be used. This is commonplace in the field of monoclonal antibodies and an ongoing preclinical study convincingly demonstrates the superior efficacy and safety of using equivalent anti-CD2. The primate study demonstrates that anti-CD2 is essential for preventing rejection, allowing cessation of chronic immunosuppression and provide improved long-term survival (abstract number 207, American Transplant Congress 2017).

Figure 3. Peripheral blood lymphocyte counts and phenotypes analyzed in chimpanzees before and after treatment with Siplizumab (humanized anti-CD2), BTI-322 (the rat counterpart), or vehicle control (normal saline). Treatments were administered day 0, 1 and 2 (indicated by arrows). ITBMed proprietary data, unpublished.

The potential of targeting the anti-CD2 pathway in clinical transplantation

The data outlined below has been generated in clinical trials of the parent rat monoclonal anti-CD2, named BTI-322, the humanization of which constitutes Siplizumab. BTI-322 thus has the same specificity as Siplizumab, differing only in its non-humanized framework, which is a common occurrence in the development of monoclonal antibodies.

Anti-CD2 treatment as induction therapy

A total of 40 patients receiving renal transplants were enrolled in a randomized study (Squifflet et al, 1997). The control group received a basic conventional triple-drug regimen. The test group received the same basic triple-drug regimen plus anti-CD2 (BTI-322) for a 10-day course at a dose of 5 mg/day.

There was a lower incidence of rejection in patients treated with anti-CD2 compared to controls (Squifflet et al, 1997), with 75% of patients treated with anti-CD2 being rejection-free compared to 40% of control patients (p = 0.009).

These promising results were subsequently confirmed also for liver transplantation (Lerut et al, 2005), where 40 patients were randomized to receive either standard-of-care alone or in combination with anti-CD2. Biopsies one week post-transplant showed significantly lower Banff (rejection) scores for patients treated with anti-CD2 compared to standard-of-care alone (2.3 vs 5.4, p < 0.0001), as well as lower incidences of moderate and severe rejection (0% vs 50%, p < 0.001). It was concluded that “treatment with anti-CD2 had a marked beneficial influence on the early histologic allograft picture, rendering early post-transplant follow-up very easy and secure”.

 

Anti-CD2 treatment in confirmed acute transplant rejections

A Phase I study was conducted in renal transplant patients undergoing acute rejection episodes (Mourad et al, 1997). Complete or partial clinical and histologic responses were obtained in 91% of the patients treated for the first episode of rejection, and in 70% of patients with an uncontrolled rejection, suggesting that anti-CD2 was a safe and effective treatment both for the first episode of acute rejection and as a rescue therapy for uncontrolled rejections.

Figure 4. Incidence of rejection in BTI-322 treated group and control group at 6 months post-transplant (p = 0.009, log rank test).

Adapted from Squifflet et al, 1997.

Siplizumab has the revolutionizing potential to induce clinical transplantation tolerance

The medical plausibility of Siplizumab is most strongly evident in a clinical trial of 10 patients that received a Siplizumab-based regimen at the time of transplantation, after which they received standard of care immunosuppression that was subsequently tapered and completely discontinued within approximately 1 year. This trial demonstrates successful immunosuppression withdrawal in the majority of patients (Figure 5), and improved safety (Figure 6) as well as improved quality of life (Figures 7 and 8), for patients treated with Siplizumab regimen (Kawai et al, 2014; Madariaga et al, 2016). In addition, there are potential cost benefits when using Siplizumab in the peri-transplant period instead of life-long use of conventional immunosuppressants.

“Therefore, treatment with a Siplizumab-based regimen in the peri-transplant period appears superior in all aspects compared to conventional treatment”

/ Erik Berglund, CEO

 

The clinical course for the three patients not demonstrated in the figure (30% of treated patients) did not differ from the standard of care with conventional immunosuppression. Specifically, one patient experienced thrombotic microangiopathy (TMA) caused by the calcineurin-inhibitors (CNI) tacrolimus, before it could be successfully weaned. This is a known side-effect of CNI and further demonstrates the detrimental effect of receiving chronic and generalized immunosuppression. Another patient was misdiagnosed before transplantation with regard to HLA-antibodies, and was in fact pre-sensitized with donor-specific antibodies. The third patient experienced an acute rejection and could not be successfully weaned off immunosuppression. Therefore, Siplizumab provided obvious benefits for 70% of treated patients with regard to prevention of rejection as well as safety (Figure 6) and quality of life (Figures 7 and 8).

Figure 5. In 7 of the 10 patients the clinical course was superior to standard of care. The table displays excellent maintained transplant results after immunosuppression discontinuation. Only beyond 5 years was mild and subclinical chronic rejection detected in 29% of patients treated with Siplizumab, prompting re-institution of low-dose standard of care immunosuppression, and no patient experienced moderate to severe chronic rejection. This is in stark contrast to the frequency of chronic rejection in patients receiving standard of care only (Nankivell et al, 2003; Cornell et al, 2005), where 94% present mild chronic rejection 1 year after transplantation (compared to 0% at 1 year in patients treated with Siplizumab) and 66% display moderate to severe chronic rejection 5 years after transplantation (compared to 0% at 5 years in patients treated with Siplizumab).

Adapted from Kawaii et al, 2014.

Figure 6. Post-transplant complications in patients receiving Siplizumab versus standard of care immunosuppression. Patients receiving Siplizumab experienced substantially less complications compared to standard of care with regard to hypertension (42% vs 82%), hyperlipidemia (0% vs 70%), diabetes (0% vs 41%), cancer (0% vs 12%) and infections (0% vs 29%). The reason for the lower risk of post-transplant complications is because treatment with Siplizumab allows minimization and cessation of standard of care immunosupppression known to cause these side-effects.

Adapted from Kawaii et al, 2014.

Figure 7. Quality of life assessed by SF-36. Five patients treated with Siplizumab (tolerant group) returned the questionnaire for assessment of quality of life and was compared to equal patients receiving standard of care (Conventional immunosuppression). Patients receiving Siplizumab had higher quality of life with regard to symptoms, burden of kidney disease, sexual function, physical function, general health, social function, energy and overall health. Thus, patients treated with Siplizumab not only experience less rejection (Figure 5) and side-effects (Figure 6), but also have improved quality of life.

Adapted from Madariaga, 2016.

Figure 8. Transplant symptom occurrence scale. Patients treated with Siplizumab experienced no symtoms to mild symptoms after transplantation with regard to depression, excessive appetite, flatulence, hearing loss, increased thirst, itching, joint pain, lack of energy, muscle cramps, reduced interest in sex and shortness of breath. These results are well in line with those obtained by SF-36 (Figure 7).

Adapted from Madariaga, 2016.

Is Siplizumab treatment safe?

Infusion-related reactions, such as chills, pyrexia, and fatigue, have been seen in patients treated with Siplizumab. These events have been mild to moderate in severity, and transient in nature. No serious adverse events and deaths related to Siplizumab have been reported when used in transplantation.