MaxCytes experiences and credentials in development

1
MaxCytes experiences and credentials in
development manufacture of DC vaccines
2
Designing effective cellular vaccinesEngineering
implications that effect clinical outcomes

• How to potentiate biological activity of cellular
  vaccines?
• Multiple desired biological characteristics for
  cancer vaccines include
• Increased survival and persistence in vivo
• Anti-apoptotic agents / genes, Akt, Factor V
• Homing to sites for effective T cell engagement
• CCL CXCR pathways for enhanced vaccine
  targeting / homing
• Enhanced ability to engage and activate effector
  T cells
• Interleukins (IL-12, IL-15, IL-23), Th-1
  polarization pathways, CD40L activation
• DC Licensing Engagement of innate immune system
  (NK cells, gd T cells)
• Downregulation of pathways that activate
  regulatory T cells
• PDL-1 / PDL-2, SOCS-1, K-DEL
• Ability to control the kinetics of antigen
  expression, processing presentation
• Control of loading, expression processing of
  antigens

3
MaxCyte technology platform enables effective
cellular vaccine development

• MOA (1) Antigen loading processing (2)
  Cellular Engineering
• Multiple desired biological characteristics for
  cancer vaccines include
• Increased survival and persistence in vivo
• Anti-apoptotic agents / genes, Akt, Factor V
• Homing to sites for effective T cell engagement
• CCL CXCR pathways for enhanced vaccine
  targeting / homing
• Enhanced ability to engage and activate effector
  T cells
• Interleukins (IL-12, IL-15, IL-23), Th-1
  polarization pathways, CD40L activation
• DC Licensing Engagement of innate immune system
  (NK cells, gd T cells)
• Downregulation of pathways that activate
  regulatory T cells
• PDL-1 / PDL-2, SOCS-1, K-DEL
• Ability to control the kinetics of antigen
  expression, processing presentation
• Control of loading, expression processing of
  antigens

4
MaxCyte technology platform enables effective
cellular vaccine development

• MOA (1) Antigen loading processing (2)
  Cellular Engineering
• Multiple desired biological characteristics for
  cancer vaccines include
• Increased survival and persistence in vivo
• Anti-apoptotic agents / genes, Akt, Factor V
• Homing to sites for effective T cell engagement
• CCL CXCR pathways for enhanced vaccine
  targeting / homing
• Enhanced ability to engage and activate effector
  T cells
• Interleukins (IL-12, IL-15, IL-23), Th-1
  polarization pathways, CD40L activation
• DC Licensing Engagement of innate immune system
  (NK cells, gd T cells)
• Downregulation of pathways that activate
  regulatory T cells
• PDL-1 / PDL-2, SOCS-1, K-DEL
• Ability to control the kinetics of antigen
  expression, processing presentation
• Control of loading, expression processing of
  antigens

Cellular Engineering
Antigen Engineering
5
Engineering Antigen Loading ProcessingExample
Tumor Lysate loaded DC vaccine

• Hypothesis
• Control of antigen uptake processing allows
  for robust enhancement of biological activity
  (POTENCY) of DC vaccine
• Value Proposition
• Robust, scalable, closed-system, regulatory
  compliant manufacturing process
• Independent of source of tumor associated
  antigens (tumor cell lysates, mRNA, exosomes,
  proteins, )
• Use of tumor cell lysates offers the unique
  advantage of patient-specific tumor material
  including patient-specific repertoire of
  personalized antigens
• Amount ( availability) of TAAs is a critical
  process constraint
• MaxCytes approach utilizes 20-40 fold lower
  amount of antigen in obtaining potency
  enhancement compared to traditional method of
  co-incubation for antigen uptake
• Impacts process economics COGS by affecting
  therapeutic dose
• Facilitate multiple administrations from single
  lot of enhanced potency vaccine

6

• Hypothesis
• Control of antigen uptake processing allows
  for robust enhancement of biological activity
  (POTENCY) of DC vaccine
• Technical Considerations (why does potency? ?)
• Electroporation does not negatively impact the
  function (potency) of dendritic cells to
  stimulate proliferation of allogeneic T cells
• Controlled loading of antigen allows for
  homogeneous antigen loading in DC, with high
  amounts of antigen being delivered into cytosolic
  compartment of DC
• Antigen delivery does not require immature DC
  (also effective in loading mature DC) therefore
  preventing antigen loss / turnover during DC
  maturation process during traditional
  co-incubation process of antigen uptake (by
  pinocytosis) into immature DC
• Cytosolic delivery enhances processing
  presentation via HLA class I pathways resulting
  in generation of robust effector T cell responses
• MaxCyte technology offers potential for
  concurrent modulation of regulatory pathways in
  DC thus overcoming impact of Treg on DC function
  in vivo leading to more robust clinical outcomes

7

• Hypothesis
• Control of antigen uptake processing allows
  for robust enhancement of biological activity
  (POTENCY) of DC vaccine
• Technical Considerations (why does potency? ?)
• Electroporation does not negatively impact the
  function (potency) of dendritic cells to
  stimulate proliferation of allogeneic T cells
• Controlled loading of antigen allows for
  homogeneous antigen loading in DC, with high
  amounts of antigen being delivered into cytosolic
  compartment of DC
• Antigen delivery does not require immature DC
  (also effective in loading mature DC) therefore
  preventing antigen loss / turnover during DC
  maturation process during traditional
  co-incubation process of antigen uptake (by
  pinocytosis) into immature DC
• Cytosolic delivery enhances processing
  presentation via HLA class I pathways resulting
  in generation of robust effector T cell responses
• MaxCyte technology offers potential for
  concurrent modulation of regulatory pathways in
  DC thus overcoming impact of Treg on DC function
  in vivo leading to more robust clinical outcomes

8
Electroporation does not adversely affect DC
function (allo-MLR capacity)

• Allogeneic T cell proliferation following DC
  stimulation, assessed by CFSE dilution (gated on
  viable CD3 cells)

• Allo-MLR was used to assess functional potency of
  electroporated mature DC to assess any (negative)
  impact of electroporation on DC function
  (allogeneic T cell stimulatory capacity)
• Electroporated mDC retain full functional
  potency, stimulating robust allo T cell expansion

31.3
No EPControl
53.0
mDC EP with tumor lysate
Percentage of viable CD3 events corresponding
to T cells that have undergone 1 round of cell
division
9

• Hypothesis
• Control of antigen uptake processing allows
  for robust enhancement of biological activity
  (POTENCY) of DC vaccine
• Technical Considerations (why does potency? ?)
• Electroporation does not negatively impact the
  function (potency) of dendritic cells to
  stimulate proliferation of allogeneic T cells
• Controlled loading of antigen allows for
  homogeneous antigen loading in DC, with high
  amounts of antigen being delivered into cytosolic
  compartment of DC
• Antigen delivery does not require immature DC
  (also effective in loading mature DC) therefore
  preventing antigen loss / turnover during DC
  maturation process during traditional
  co-incubation process of antigen uptake (by
  pinocytosis) into immature DC
• Cytosolic delivery enhances processing
  presentation via HLA class I pathways resulting
  in generation of robust effector T cell responses
• MaxCyte technology offers potential for
  concurrent modulation of regulatory pathways in
  DC thus overcoming impact of Treg on DC function
  in vivo leading to more robust clinical outcomes

10
More DC are loaded with Antigen Greater amount
of Antigen per cell

• Requires less antigen and permits flexible DC
  loading with multiple antigens (protein, lysate,
  mRNA, DNA) ? lower COGS improved antigenic
  diversity
• Enhanced antigen loading
• More DC are loaded with antigen
• There is greater amount of antigen present per
  cell
• Electroporation can be used to load antigens into
  both immature DC (imDC) and into mature DC (mDC)
• Antigen loading into mDC (versus imDC is
  preferred because it) leads to enhanced
  antigen-specific T cell expansion(co-incubation
  is hypothesized to lead to antigen loss during ex
  vivo DC maturation)
• Direct loading into cytoplasm results in
  generation of potent effector T cell response
  (CTL)
• Molecules in addition to antigen can be loaded
  into DC to modulate effector function and
  downregulate pathways involved in Treg
  activationCellular Engineering

11
imDC mDC can be Antigen-loadedOnly imDC uptake
antigen (pinocytosis) during co-incubation

• Requires less antigen and permits flexible DC
  loading with multiple antigens (protein, lysate,
  mRNA, DNA) ? lower COGS improved antigenic
  diversity
• Enhanced antigen loading
• More DC are loaded with antigen
• There is greater amount of antigen present per
  cell
• Electroporation can be used to load antigens into
  both immature DC (imDC) and into mature DC (mDC)
• Antigen loading into mDC (versus imDC is
  preferred because it) leads to enhanced
  antigen-specific T cell expansion(co-incubation
  is hypothesized to lead to antigen loss during ex
  vivo DC maturation)
• Direct loading into cytoplasm results in
  generation of potent effector T cell response
  (CTL)
• Molecules in addition to antigen can be loaded
  into DC to modulate effector function and
  downregulate pathways involved in Treg
  activationCellular Engineering

12
SUMMARY Value proposition in the enablement of
DC vaccine therapy

• Requires less antigen and permits flexible DC
  loading with multiple antigens (protein, lysate,
  mRNA, DNA) ? lower COGS improved antigenic
  diversity
• Enhanced antigen loading
• More DC are loaded with antigen
• There is greater amount of antigen present per
  cell
• Electroporation can be used to load antigens into
  both immature DC (imDC) and into mature DC (mDC)
• Antigen loading into mDC (versus imDC is
  preferred because it) leads to enhanced
  antigen-specific T cell expansion(co-incubation
  is hypothesized to lead to antigen loss during ex
  vivo DC maturation)
• Direct loading into cytoplasm results in
  generation of potent effector T cell response
  (CTL)
• Molecules in addition to antigen can be loaded
  into DC to modulate effector function and
  downregulate pathways involved in Treg
  activationCellular Engineering

13
Electroloading of mature DC elicits superior T
cell expansion compared to immature DC
mg lysate/1 x 106 cells
mg lysate/1 x 106 cells
0.01 0.04
0.01 0.04
0.86
0.27
0.17
1.54
EP of Immature DC
EP of Mature DC
14

• Hypothesis
• Control of antigen uptake processing allows
  for robust enhancement of biological activity
  (POTENCY) of DC vaccine
• Technical Considerations (why does potency? ?)
• Electroporation does not negatively impact the
  function (potency) of dendritic cells to
  stimulate proliferation of allogeneic T cells
• Controlled loading of antigen allows for
  homogeneous antigen loading in DC, with high
  amounts of antigen being delivered into cytosolic
  compartment of DC
• Antigen delivery does not require immature DC
  (also effective in loading mature DC) therefore
  preventing antigen loss / turnover during DC
  maturation process during traditional
  co-incubation process of antigen uptake (by
  pinocytosis) into immature DC
• Cytosolic delivery enhances processing
  presentation via HLA class I pathways resulting
  in generation of robust effector T cell responses
• MaxCyte technology offers potential for
  concurrent modulation of regulatory pathways in
  DC thus overcoming impact of Treg on DC function
  in vivo leading to more robust clinical outcomes

15
Cytosolic Antigen delivery into DC
?Antigen-specific effector response

• Requires less antigen and permits flexible DC
  loading with multiple antigens (protein, lysate,
  mRNA, DNA) ? lower COGS improved antigenic
  diversity
• Enhanced antigen loading
• More DC are loaded with antigen
• There is greater amount of antigen present per
  cell
• Electroporation can be used to load antigens into
  both immature DC (imDC) and into mature DC (mDC)
• Antigen loading into mDC (versus imDC is
  preferred because it) leads to enhanced
  antigen-specific T cell expansion(co-incubation
  is hypothesized to lead to antigen loss during ex
  vivo DC maturation)
• Direct loading into cytoplasm results in
  generationof potent effector T cell response
  (CTL)
• Molecules in addition to antigen can be loaded
  into DC to modulate effector function and
  downregulate pathways involved in Treg
  activationCellular Engineering

16
Significantly higher antigen-specific T cell
expansion at 40-fold lower antigen amount
Co-incubation 8.0mg/mL
EP
EP
EP
2.0mg/mL
0.5mg/mL
8.0mg/mL
Tetramer
Antigen
1.53
0.67
3.35
3.00
HLA-A2-Antigen Tetramer-PE
Negative
0
0
0
0
Anti-CD8-FITC
17

• Hypothesis
• Control of antigen uptake processing allows
  for robust enhancement of biological activity
  (POTENCY) of DC vaccine
• Technical Considerations (why does potency? ?)
• Electroporation does not negatively impact the
  function (potency) of dendritic cells to
  stimulate proliferation of allogeneic T cells
• Controlled loading of antigen allows for
  homogeneous antigen loading in DC, with high
  amounts of antigen being delivered into cytosolic
  compartment of DC
• Antigen delivery does not require immature DC
  (also effective in loading mature DC) therefore
  preventing antigen loss / turnover during DC
  maturation process during traditional
  co-incubation process of antigen uptake (by
  pinocytosis) into immature DC
• Cytosolic delivery enhances processing
  presentation via HLA class I pathways resulting
  in generation of robust effector T cell responses
• MaxCyte technology offers potential for
  concurrent modulation of regulatory pathways in
  DC thus overcoming impact of Treg on DC function
  in vivo leading to more robust clinical outcomes

18
Regulation of Dendritic Cell FunctionThe ? of
Immune Activation v/s Immunosuppression
PD-1
SOCS-1
SOCS Suppressor of Cytokine Stimulation PD-1
Programmed Death 1
From Gilboa E, Nature Biotechnology 22 1521,
2004
19
SUMMARY Value proposition in the enablement of
DC vaccine therapy

• Requires less antigen and permits flexible DC
  loading with multiple antigens (protein, lysate,
  mRNA, DNA) ? lower COGS improved antigenic
  diversity
• Enhanced antigen loading
• More DC are loaded with antigen
• There is greater amount of antigen present per
  cell
• Electroporation can be used to load antigens into
  both immature DC (imDC) and into mature DC (mDC)
• Antigen loading into mDC (versus imDC is
  preferred because it) leads to enhanced
  antigen-specific T cell expansion(co-incubation
  is hypothesized to lead to antigen loss during ex
  vivo DC maturation)
• Direct loading into cytoplasm results in
  generation of potent effector T cell response
  (CTL)
• Molecules in addition to antigen can be loaded
  into DC to modulate effector function and
  downregulate pathways involved in Treg
  activationCellular Engineering

20

• Hypothesis
• Control of antigen uptake processing allows
  for robust enhancement of biological activity
  (POTENCY) of DC vaccine
• Pre-Clinical Proof-of-Concept (potency?)
• In vitro data (rodent model comparison with
  co-incubation loaded DC)
• Enhanced Th-1 profile (IFN-g secretion) following
  T cell stimulation
• Stimulated expanded T cells exhibit increased
  ability to recognize and kill tumor cells in
  vitro
• In vivo data (rodent model comparison with
  co-incubation loaded DC)
• Increased efficacy in tumor challenge model
• Effectiveness in therapeutic vaccination model
  (statistically significant inhibition of growth
  of pre-established tumor)
• Translation to human in vitro system and
  subsequent scale-up to robust, closed system,
  regulatory-compliant manufacturing process

21
Tumor lysate-loaded DC vaccineJ Immunotherapy,
2005

• In vitro function (Tumor lysate loaded APC)

• Tumor Challenge Model

• Therapeutic Vaccination Model

22
Process Optimization ObjectivesConsistency ?,
Potency ?, COGS ?, Mfgng ?

• Current Process

• MaxCyte Process

Integration with Upstream Process(for example
COBE Elutra) Potential for reduced Cell Culture
TimeReduced (20-40X) Tumor Lysate
needConsistent processing (Quality) Reduced
Overall Culture Duration(Antigen Loading 1
hour versus 1 day) Improved POTENCYMultiple
Lots per LeukapheresisPotential for improved
OUTCOME Seamless transfer to cGMP at CMO
23
MaxCyte technology ? Enhanced potency for DC
vaccine compared to co-incubation
24
MaxCytes Technology has been already validated
as value-add for DC vaccines

• MaxCyte has demonstrated the value proposition of
  its cell-loading technology platform in the
  development and manufacture of antigen presenting
  cell based vaccines
• MaxCyte technology results in enhancement of
  potency (tumor-antigen specific T cell
  stimulation) of APC vaccines
• MaxCyte has entered into Clinical / Commercial
  License and Supply Agreement for Licensing of
  MaxCytes technology for development of cancer
  vaccine products
• Geron (US) Telomerase mRNA loaded DC vaccine
• Medinet (JP) Immuno-cell Therapy Product

25
Value proposition for enhancing potency of DC
products

• High viability recovery (yield / dose) of
  cells
• Efficient loading with 20-40 fold lower
  requirement for Tumor lysate
• Significantly enhanced potency (T cell
  activation in vitro)
• Closed system for aseptic cGMP operations
• Consistent, Robust, Scalable process (Identity,
  Purity Potency)
• Significant impact on COGS for manufacture of
  patient-specific lots
• Potential for (future) enhancements in
  controlling Treg effects
• Enhanced potency no additional risk (compared
  to co-incubation)